Sample manipulation and assay with rapid temperature change

ABSTRACT

Among other things, the present invention is related to devices and methods of performing biological and chemical assays, particularly an easy sample manipulation and/or a rapid change or a rapid thermal cycling of a sample temperature is needed (e.g. Polymerase Chain Reaction (PCR) for amplifying nucleic acids).

CROSS-REFERENCING

This application is a National Stage entry (§ 371) application ofInternational Application No. PCT/US18/65297, filed on Dec. 12, 2018,which claims the benefit of U.S. Provisional Patent Application No.62/597,851, filed on Dec. 12, 2017, U.S. Provisional Patent ApplicationNo. 62/772,597, filed on Nov. 28, 2018, International Application No.PCT/US2018/017307, filed on Feb. 7, 2018, International Application No.PCT/US2018/018108, filed on Feb. 14, 2018, International Application No.PCT/US2018/018405, filed on Feb. 15, 2018, International Application No.PCT/US2018/028784, filed on Apr. 23, 2018, and International ApplicationNo. PCT/US2018/034230, filed on May 23, 2018, the contents of which arerelied upon and incorporated herein by reference in their entirety.

The entire disclosure of any publication or patent document mentionedherein is entirely incorporated by reference.

FIELD

Among other things, the present invention is related to devices andmethods of performing biological and chemical assays, particularly aneasy sample manipulation and/or a rapid change or a rapid thermalcycling of a sample temperature is needed (e.g. Polymerase ChainReaction (PCR) for amplifying nucleic acids).

BACKGROUND

In certain chemical, biological, or medical assays, an easy samplemanipulation and/or a rapid change or a rapid thermal cycling of asample temperature is needed (e.g. Polymerase Chain Reaction (PCR) foramplifying nucleic acids).

In certain situation, one would like to quickly isolate a part of thefluidic sample from the rest to do analysis. During thermal cycling, aliquid sample will change in its volume with temperature, and this cancause liquid sample flow between the sample area being heated and thesample area not being heated. Such liquid sample flow can change thesample temperature and increase the time and energy needed to do thermalcycling. Therefore, there is a need to reduce the liquid sample flowduring thermal cycling.

One objective of the present invention is to address how to quicklyisolate a part of the fluidic sample from the rest to do analysis.

Another objective of the present invention is to address the need toreduce the liquid sample flow between two different temperature areasduring thermal cycling. The present invention additionally providesdevices and methods for isothermal nucleic acid amplification.

SUMMARY OF INVENTION

The following brief summary is not intended to include all features andaspects of the present invention.

The present invention provides, among other things, the devices andmethods that can rapidly change or cycle (i.e. heating and cooling) asample temperature with high speed, less heating energy, high energyefficiency, a compact and simplified apparatus (e.g. handheld), easy andfast operation, and/or low cost.

The present invention has experimentally achieved a cycling of a sampletemperature between 95° C. and 55° C.) in a second or less.

The invention has six novel aspects (1) the devices and methods thatallow fast thermal cycling, (2) the devices and methods that allow thesample thickness uniform and sample holder mechanically stable forhandling, (3) simple operation, (3) devices and methods for doing realtime PCR (4) biochemistry, and (5) smartphone based systems.

To rapid thermal cycle the temperature of a sample or a portion of it,one must reduce the thermal mass and lateral heat.

Radiative heating and cooling are preferred.

BRIEF DESCRIPTION OF THE DRAWINGS

The skilled artisan will understand that the drawings, described below,are for illustration purposes only. The drawings are not intended tolimit the scope of the present teachings in any way. In some cases, thedrawings are not in scale. In the figures that present experimental datapoints, the lines that connect the data points are for guiding a viewingof the data only and have no other means.

FIG. 1A shows a sectional view of an embodiment of the system of thepresent invention, comprising a sample holder and a clamping structure.The active clamp. Panel (A) illustrates the system before the clampingstructure is activated, where the two rings of the clamp are open andhence does not assert a force to push the two plate together. Panel (B)illustrates that the clamp is activated, where a force is applied by theclamping structure to pinch the sample holder area that is pressed bythe claim.

FIG. 1B shows a top view of an embodiment of one side of a ring clamp.

FIG. 2 shows a sectional view of an embodiment of the system of thepresent invention, comprising a sample holder, and a clamping structure.Panel (A) illustrates the system before the ring clamping structure isactivated, where the two rings of the clamp does not assert a force topush the two plate together. Panel (B) illustrates the system after aforce is applied by the clamping structure.

FIG. 3 . shows a sectional view of an embodiment of the system of thepresent invention, comprising a sample holder and a clamping structure.Panel (A) illustrates the system before the clamping structure isactivated. Panel (B) illustrates the system after a force is applied bythe clamping structure.

FIG. 4 . shows a sectional view of an embodiment of the system of thepresent invention, comprising a first plate, a second plate with a welland spacers that are fixed on the inner surface, and a clampingstructure. Panel (A) illustrates the system before the clampingstructure is activated. Panel (B) illustrates the system after a forceis applied by the clamping structure.

FIG. 5 . shows exemplary embodiments of two types of clampingstructures. Panel (A) comprises a support with a one-spring ringstructure. Panel (B) comprises a support with a four-spring ringstructure.

FIG. 6 shows a schematic illustration of certain components of a systemfor changing the temperature of a sample and for monitoring a signalfrom the sample, according to some embodiments.

FIG. 7A shows an embodiment of a device with a heating layer separatedfrom a cooling layer, according to some embodiments.

FIG. 7B shows an embodiment having a heating layer contacting a coolinglayer, according to some embodiments. FIG. 7C schematically illustratesa perspective view of an embodiment of a device with a heating/coolinglayer, in accordance with an embodiment. FIG. 7D schematicallyillustrates a sectional view of an embodiment of a device with

FIGS. 8A and 8B show a prospective view and a sectional view,respectively, of a combination heating/cooling layer on an outer surfaceof a plate, according to some embodiments.

FIG. 9A shows perspective and sectional views of the device in an openconfiguration, according to some embodiments.

FIG. 9B shows perspective and sectional views of the device when thesample holder is in a closed configuration, according to someembodiments.

FIG. 10 shows a sectional view of a system showing additional elementsthat facilitate temperature change and control, according to someembodiments.

FIG. 11A schematically illustrates a system of heating and temperaturemonitoring device for the assay device, according to some embodiments.

FIG. 11B schematically illustrates a field of view of the thermal imagerin the system described in FIG. 11A when heating up the assay device,according to some embodiments.

FIG. 12 schematically illustrates a real-time PCR system comprising aheating source and temperature monitoring system as described FIG. 11Aand FIG. 11B and a pair of fluorescent excitation light source anddetector, according to some embodiments.

FIG. 13 schematically illustrates an embodiment of a real-time PCRsystem comprising a heating source, a fan and temperature detector as atemperature control system and a pair of fluorescent excitation lightsource (with filter) and detector (with lens and filter) as the realtime detection system.

FIG. 14 (a) shows a perspective view of a round heating tube and ahexagonal heating tube with a diameter of 6 mm and a point LED lightsource at the center of one tube end, according to some embodiments.

FIG. 14 (b) shows the optical beam intensity measured at the other endof tube, according to some embodiments.

FIG. 15 shows a working SNAP PCR amplification of nucleic acid (E-coliplasmid DNA) with assay device demonstrating (a) 4.5 sec thermal cyclingtime (1 sec heating time from 60° C. to 95° C., 0.5 sec staying at 95°C., 2.5 sec cooling time from 95° C. to 60° C., and 0.5 sec staying at60° C.); (b) Gel electrophoresis results of SNAP PCR products ran in 3minutes (40 cycles) and conventional PCR products (40 cycles) ran in 40minutes shows 3 min SNAP PCR has a comparable amplification performanceas 40 min conventional PCR. The M line in the figure is a Gelelectrophoresis marker with 100 bp line marked. Both SNAP PCR andconventional PCR have clear 100 bp production line and similarintensity. Negative sample without template does not show bar in gelanalyze

FIG. 16 show a sectional view of an embodiment of the system of thepresent invention, comprising a sample holder and a clamping structure.FIG. 16A illustrates the system before the clamping structure isactivated, where the two rings of the clamp are open and hence do notexert a force to push the two plates together. FIG. 16(B) illustratesthat the clamp is activated, where a force is applied by the clampingstructure to pinch the sample holder area that is pressed by the clamp.

FIG. 17 shows a top view of an embodiment of one side of a ring clamp.

FIG. 18 shows a sectional view of an embodiment of the system of thepresent invention, comprising a sample holder, and a clamping structure.The sample holder comprises a first plate and a second plate that aremovable to each other, wherein the second plate a well. FIG. 18Aillustrates the system before the ring clamping structure is activated,where the two rings of the clamp do not assert a force to push the twoplates together. FIG. 18B illustrates the system after a force isapplied by the clamping structure.

FIG. 19 . shows a sectional view of an embodiment of the system of thepresent invention, comprising a sample holder and a clamping structure.The sample holder comprises a first plate, a second plate with spacersthat are fixed on the inner surface. FIG. 19A) illustrates the systembefore the clamping structure is activated. FIG. 19B illustrates thesystem after a force is applied by the clamping structure.

FIG. 20 . shows a sectional view of an embodiment of the system of thepresent invention, comprising a first plate, a second plate with a welland spacers that are fixed on the inner surface, and a clampingstructure. FIG. 20A illustrates the system before the clamping structureis activated. FIG. 20B illustrates the system after a force is appliedby the clamping structure.

FIG. 21 schematically illustrates two types of clamping structures. FIG.21A comprises a support with a one-spring ring structure. FIG. 21Bcomprises a support with a four-spring ring structure.

FIG. 22 schematically illustrates certain components of a system forchanging the temperature of a sample and for monitoring a signal fromthe sample, according to some embodiments.

FIG. 23A schematically illustrates a device with a heating layerseparated from a cooling layer, in accordance with one or moreembodiments.

FIG. 23B schematically illustrates an embodiment having a heating layercontacting a cooling layer.

FIGS. 24A and 24B schematically illustrate a prospective view and asectional view, respectively, of a combination heating/cooling layer onan outer surface of a plate, in accordance with one or more embodiments.

FIG. 25A schematically illustrates perspective and sectional views ofthe device in an open configuration, in accordance with one or moreembodiments.

FIG. 25B schematically illustrates perspective and sectional views ofthe device when the sample holder is in a closed configuration, inaccordance with one or more embodiments.

FIG. 26 schematically illustrates a top view of a device, in accordancewith one or more embodiments.

FIG. 27A schematically illustrates the perspective view of the systemwhen the device (sample holder of the system) is in an openconfiguration, in accordance with one or more embodiments.

FIG. 27B schematically illustrates a sectional view of the system whenthe sample holder is in a closed configuration, in accordance with oneor more embodiments.

FIG. 28 schematically illustrates a sectional view of a system showingadditional elements that facilitate temperature change and control,according to some embodiments.

FIGS. 29A and 29B schematically illustrates a perspective view and asectional view, respectively, of the device having multiple samplecontact areas, according to some embodiments.

FIG. 30 schematically illustrates sectional views of a device,demonstrating how the sample is added and compressed, in accordance withone or more embodiments.

FIG. 31 schematically illustrates sectional views of a device,demonstrating a PCR process, in accordance with one or more embodiments.

FIGS. 32A and 32B schematically illustrates a top view and a sectionalview, respectively, of a heating layer on a plate of the device, inaccordance with one or more embodiments.

FIGS. 33A and 33B schematically illustrates sectional views of a devicehaving a first plate, a second plate, and a heating/cooling layer, inaccordance with some embodiments.

FIG. 34 schematically illustrates a sectional view of a system torapidly change the temperature of a sample, including a heating sourceusing a fiber, in accordance with one or more embodiments.

FIG. 35 schematically illustrates a sectional view of a system torapidly change the temperature of a sample, including a heating sourceusing a lens, in accordance with one or more embodiments.

FIGS. 36A and 36B schematically illustrate a top view and side view,respectively, of a device having a separate heating element, inaccordance with one or more embodiments.

FIGS. 37A and 37B schematically illustrate a perspective view and a sideview, respectively, of an optical pipe used to guide electromagneticwaves (e.g., light) from a heating source, in accordance with one ormore embodiments.

FIG. 38 schematically illustrates a perspective view of an optical pipe,in accordance with one or more embodiments.

FIGS. 39A and 39B schematically illustrate a side view and a top view,respectively, a sample device that is heated with a heat source, inaccordance with one or more embodiments.

FIG. 40 schematically illustrates a schematic side view of the device,having a lens that focuses light from a heat source, in accordance withone or more embodiments.

FIG. 41 shows experimental absorption spectra of different materials, inaccordance with an embodiment.

FIG. 42 shows experimental thermal cycling data, in accordance with anembodiment.

FIG. 43 shows experimental data of the effects of the area of theheating/cooling layer on heating and cooling time, in accordance with anembodiment.

FIG. 44 shows experimental data of the heating and cooling time vs. thearea size of the heating/cooling layer, in accordance with anembodiment.

FIG. 45A shows experimental data of the relationship between the heatingtime and the heating/cooling layer thickness, in accordance with anembodiment.

FIG. 45B shows experimental data of the relationship between the coolingtime and the heating/cooling layer thickness, in accordance with anembodiment.

FIG. 46A shows experimental data of the relationship between the heatingtime and the distance between the heating/cooling layer and the sample,in accordance with an embodiment.

FIG. 46B shows experimental data of the relationship between the coolingtime and the distance between the heating/cooling layer and the sample,in accordance with an embodiment.

FIG. 47A shows experimental data of the relationship between the heatingtime and the sample layer thickness, in accordance with an embodiment.

FIG. 47B shows experimental data of the relationship between the coolingtime and the sample layer thickness, in accordance with an embodiment.

FIG. 48A shows experimental data of the relationship between the heatingtime and the heating source power, in accordance with an embodiment.

FIG. 48B shows experimental data of the relationship between the coolingtime and the heating source power on the sample, according to someembodiments.

FIG. 49A shows experimental data of the relationship between the heatingtime and different heating/cooling layer materials, according to someembodiments.

FIG. 49B shows experimental data of the relationship between the coolingtime and different heating/cooling layer materials, according to someembodiments.

FIG. 50A shows a schematic of a device having sphere-shaped spacers,according to some embodiments.

FIG. 50B shows a schematic of a device having pillar-like spacers,according to some embodiments.

FIGS. 51A and 51B show a top view and a side view, respectively, of adevice on a support, according to some embodiments.

FIG. 52 shows experimental data of the effects of putting a device on adevice support and/or a device adaptor on heating and cooling time,according to some embodiments.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

The following detailed description illustrates some embodiments of theinvention by way of example and not by way of limitation. If any, thesection headings and any subtitles used herein are for organizationalpurposes only and are not to be construed as limiting the subject matterdescribed in any way. The contents under a section heading and/orsubtitle are not limited to the section heading and/or subtitle, butapply to the entire description of the present invention.

The citation of any publication is for its disclosure prior to thefiling date and should not be construed as an admission that the presentclaims are not entitled to antedate such publication by virtue of priorinvention. Further, the dates of publication provided can be differentfrom the actual publication dates which can need to be independentlyconfirmed.

Definitions

The term “sample thermal cycler” or “thermal cycler” refers an apparatusthat can raise and cool temperature of a sample, and can, if needed, torepeatedly heat and cool a sample between two temperatures.

The term “sample thermal cycling” or “thermal cycling” refers to arepeatedly raising and cooling temperature of a sample.

The term “a sample thermal cycle” or “a thermal cycle” refers to a cyclethat raises the sample temperature to a higher temperature and then coolit back to the original temperature.

The term “sample thermal cycling time” or “thermal cycling time” refersthe time for performing a given numbers of thermal cycle.

The term “sample thermal cycling speed” or “thermal cycling speed”refers the speed for performing thermal cycle.

The term “thermal mass” of a material refers to the energy needed toheat up the temperature of that material by one degree when there is noother energy loss. Hence the thermal mass of a material is equal to thespecific heat per unit volume multiplies the volume of the material.

The term “thermal conductivity-to-capacity ratio” refers the ratio ofthe thermal conductivity of a material to its thermal capacity. Forexamples, at about room temperature, the thermalconductivity-to-capacity ratio is 1.25 cm{circumflex over ( )}2/sec(centimeter-square/second) for gold and 1.4×10⁻³ cm{circumflex over( )}2/sec for water.

The term “wasted energy” refers the energy supplied to a sample holderthat is not used to directly heat the relevant sample.

The terms “a” and “an”, as used herein, unless clearly indicated to thecontrary, should be understood to mean “at least one”.

The term “about,” as used herein, generally refers to a range that is15% greater than or less than a stated numerical value within thecontext of the particular usage. For example, “about 10” would include arange from 8.5 to 11.5.

The term “sample holder support” refers to a device that a sample holderis physically attached to the device and mechanically supported by thedevice.

The term “disposable”, as used herein, generally refers to devices whichare designed to be discarded after a limited use (e.g., in terms ofnumber of reactions, thermal cycles, or time) rather than being reusedindefinitely.

The term “nucleic acid amplification” refers to the production of one ormore replicate copies of an existing nucleic acid.

The term “nucleic acid amplification cycle” refers to a complete set ofsteps used to perform a single round of nucleic acid amplification.

The term “template” refers to a nucleic acid that is amplified.

The term “amplification product” refers to replicate copies of anexisting nucleic acid produced during nucleic acid amplification from atemplate.

The term “black paint” refers to a paint that has a black color to humaneye when under a day light illumination.

The term “cooling gas” or “cooling liquid” refers to a gas or liquidphase, respectively, which is used to remove thermal energy, forexample, from a sample, from a sample holder, from a material, or from aregion.

The term “mechanical contact”, as used herein, generally refers tocontact made between one or more materials wherein the materials arephysically touching.

The term “thermal path” refers to the distance through which thermalenergy transfers from one location to another location.

The term “relevant sample” or “relevant sample volume” refers to thevolume of the sample that is being heated and/or cooled to desiredtemperatures during a thermal cycling, and the relevant sample can be aportion or an entire volume of a sample on a sample holder, and there isno fluidic separation between the a portion of the sample to the rest ofthe sample.

The term “high-K material” refers to a material that has a thermalconductivity (K) equal to or larger than 50 W/(m·K) (e.g. gold: ˜314W/(m·K) and graphite ˜80 W/(m·K) are high-K material).

The term “low-K material” refers to a material that has a thermalconductivity (K) equal to or less than 1 W/(m·K) (e.g. water (˜0.6W/(m·K)) and plastic (˜0.2 W/(m·K)) are low-K material).

The terms “cooling time in a thermal cycle” and “cooling cycle time” areinterchangeable.

The terms “heating time in a thermal cycle” and “heating cycle time” areinterchangeable.

The term “heating zone” refers to (a) the heating layer when the heatinglayer is a separate layer from the cooling layer; or (b) the area ofheating when the heating and the cooling use the same layer; the heatingzone is being directly heated by a heating source.

The term “directly heated” means that an energy being put into thatarea. For example, for a heating zone by a LED heating source, the LEDheating source projects a light over the heating zone. For a heatingzone by an electrical heating source, the electrical hearing sourcesends an electrical current to the heating zone to create heat in theheating zone.

The term “cooling zone” refers to (a) the cooling layer when the coolinglayer is a separate layer from the heating layer; or (b) the area ofcooling when the cooling and the heating use the same layer. A coolingzone, unless stated otherwise, comprises a material of a thermalconductivity of 50 W/m- or larger.

The term “a heating layer is heated by a heating source” means that “aheating layer or a heating zone of a heating/cooling layer is heated aheating source”.

The term “non-sample material” refers to the materials on a sampleholder that are outside the relevant sample volume.

The term “wasted heating energy” refers to the energy that must besupplied to the non-sample materials and the non-relevant samples, inorder to heat the relevant sample volume to a desired temperature.

The term “average linear dimension” of an area is defined as a lengththat equals to the area times 4 then divided by the perimeter of thearea. For example, the area is a rectangle, that has width w, and lengthL, then the average of the linear dimension of the rectangle is4*W*L/(2*(L+W)) (where “*” means multiply and “/” means divide). By thisdefinition, the average line dimension is, respectively, W for a squareof a width W, and d for a circle with a diameter d.

The term “lateral” refers to the direction that is parallel to theplates of a sample holder.

The term “vertical” refers to the direction that is normal to the platesof a sample holder.

The term “period” of periodic structure array refers to the distancefrom the center of a structure to the center of the nearest neighboringidentical structure.

The term “smart phone” or “mobile phone”, which are usedinterchangeably, refers to the type of phones that has a camera andcommunication hardware and software that can take an image using thecamera, manipulate the image taken by the camera, and communicate datato a remote place. In some embodiments, the Smart Phone has a flashlight.

The term “heating layer”, or “heating zone”, unless stated otherwise,refers to a material layer that comprises at least a layer of a materialthat has a thermal conductivity of 50 W/m-K or larger.

The term “heating volume” refers to the volume of a material to beheated.

The term “cooling layer” refers to a thermal radiative cooling layerwith a high thermal conductivity and has a large surface thermalradiation capability that is at least 50% of that of a blackbody.

The term “lateral dimension” or “lateral area” of the sample inside thesample holder for heating and cooling, refers lateral dimension orlateral area of the portion of the sample that is being heated to adesired temperature.

The term “plate” refers to a plate this is free standing, except thatwhen two plates are in a “closed configuration” where the two plates areclose together and separated by spacers (in this case the pair of theplates are free standing). The term of “free standing” means that thecenter region of the plate is free of any support. For example, when twoplates are in a closed configuration and the sample is between theplate. The central region of the plate pair has no mechanical support,only air touches the outside surface of the plates.

The term “clamp” and “clamping structure” are used interchangeableherein.

I. Sample Manipulation and Assay with Rapid Temperature Change

The present invention provides, among other things, devices and methodsto (a) quickly isolate a part of the fluidic sample from the rest to doanalysis, and (b) improve the time and energy needed in thermal cyclingof a liquid sample by reducing the flow of the liquid sample from theinside to the outside of a thermal cycling sample area.

In certain chemical, biological, or medical assays, a rapid change or arapid thermal cycling of a sample temperature is needed (e.g. Polymerasechain reaction (PCR) for amplifying nucleic acids).

During thermal cycling, a liquid sample will change in its volume withtemperature, and this can cause liquid sample flow. Liquid sample flowcan change the sample temperature and increase the time and energyneeded to do thermal cycling. Therefore, there is a need to reduce theliquid sample flow during thermal cycling.

One objective of the present invention is to address the need to reducethe liquid sample flow during thermal cycling. The present inventionadditionally provides devices and methods for isothermal nucleic acidamplification.

A. Quickly Isolating a Portion of a Fluidic Sample

In certain embodiments of the present invention, a device forfluidically isolating a portion of a sample, comprising a first plate, asecond plate, and a clamp, wherein:

-   -   i. wherein one or both of the plates is flexible, wherein the        plates sandwich a fluidic sample to be analyzed that has a        thickness 200 um or less, and has a sample area at last 100        times larger than the sample thickness; and    -   ii. the clamp comprises two jaws comprising a top ring and a        bottom ring that are movable to each other; and    -   iii. the clamp has two operation modes:

(a) a non-active mode, wherein the top ring and the bottom ring of theclamp do not push the first plate and second plate together; and

(b) an active mode, wherein the top ring and the bottom ring of theclamp apply force to squeeze the first plate and the second plate anddeform the area of the flexible plates that is under the compression ofthe clamp, thereby reducing the spacing between the two plates in thatarea, and wherein the reduction of the plate spacing reduces or preventsa fluidic flow between a sample portion encircled by the rings and asample portion outside the rings.

In certain embodiments of the present invention, device for fluidicallyisolating a portion of a sample, comprising a first plate, a secondplate, spacers, and a clamp, wherein:

-   -   i. the first plate and the second plate are movable relative to        each other into different configurations, including an open        configuration and a closed configuration, wherein one or both of        the plates is flexible, wherein each of the plates comprises, on        its respective surface, a sample contact area for contacting a        fluidic sample, and wherein at a closed configuration the plates        sandwich a sample to be analyzed, that has a thickness 200 um or        less, and has an sample area at last 100 times larger than the        sample thickness;    -   ii. the spacers have a predetermined substantially uniform        height that is equal to or less than 200 microns, wherein at        least one of the spacers is inside the sample contact area; and    -   iii. the clamp has two operation modes:

(a) a non-active mode, wherein the top ring and the bottom ring of theclamp do not push the first plate and second plate together; and

(b) an active mode, wherein the top ring and the bottom ring of theclamp are configured to insert force to squeeze the first plate and thesecond plate and deform the area of the flexible plates that is underthe compression of the clamp, thereby reducing the spacing between thetwo plates in that area, and wherein the reduction of the plate spacingreduces or prevents a fluidic flow between a sample portion encircled bythe rings and a sample portion outside the rings.

wherein in the open configuration the two plates are partially orcompletely separated apart, the spacing between the plates is notregulated by the spacers, and the clamp is in non-active mode, and thesample is deposited on one or both of the plates;

wherein in the closed configuration, at least a part of the sampledeposited in the open configuration is compressed by the two plates intoa layer of substantially uniform thickness, wherein the uniformthickness of the layer is regulated by the plates and the spacers.

One advantage of using a ring (i.e. encircled shape) is that once therings are clamped on, due to a constant volume of the sample liquid,even the plate thickness is very thin (e.g. 20 um), the two plates willhelp keeping the spacing of the two plates constant.

Some of the principles of a clamp are shown in the figures. As show, theclamp has two operation modes: a non-active mode and an active mode.When the plates are in the open configuration the clamp is in thenon-active mode. When the plates are in the closed configuration and theclamp is activated, the clamp is in the active mode.

In some embodiments, activation of the clamp reduces flow from inside ofthe clamp ring to outside of the clamp ring. As such, the clamp can beused to create an at least partially isolated reaction chamber in thedevice. In some embodiments, the device may contain several clamp rings,each capable of creating a separate reaction chamber. In someembodiments, each ring as multiple of smaller diameter rings, thatisolate a sample into multiple pockets. For example, in someembodiments, a device may contain at least 2, at least 4, at least 8, atleast 16, at least 32 or at least 64 or more top and bottom clamp rings,where the top rings are movable relative to the bottom rings and, whenthe plates are in the closed position, the rings oppose each other andpinch different areas of the device to produce multiple isolatedreaction chambers.

The rings may be of any shape, e.g., circular, oval, rectangular,pentagonal, hexagonal, square, star, or any combination thereof withoptional rounded corners. In some embodiments, the clamp ring has ashape of circular, elliptical, oval, rectangular, pentagonal, hexagonal,square, star, polygon, or any superposition of these shapes. In someembodiments, the clamp ring has a preferred shape of circular,elliptical, oval, or any superposition of these shapes.

The parts of the clamp that are in contact with the plate (the rings ofthe clamp) may have a cross-section of any shape and may be e.g., round,square, triangular, or rectangular with optional rounded corners, forexample. As noted below, in some embodiments, the cross-section of thetop ring may be of a different shape to the bottom ring. For example, insome embodiments, one of the rings may have a sharp edge that is contactwith one of the plates and the other ring may have a flat area incontact with the plates. In this configuration, activation of the clampmay crush the spacers between the rings, thereby creating an at leastpartially isolated reaction chamber.

The shape and dimensions of the area defined by the rings, i.e., the“ring area” or the area in the interior of the rings, depends on theshape and dimensions of the rings and, as such, may vary greatlydepending on how the device is implemented. In some embodiments, thering area may be less than 10,000 mm², less than 5,000 mm², less than3,000 mm², less than 1000 mm², less than 500 mm², less than 300 mm²,less than 100 mm², less than 50 mm², less than 20 mm², less than 10 mm²,less than 5 mm², less than 2 mm², less than 1 mm², less than 0.5 mm² orless than 0.1 mm². The term “ring area” is intended to refer to an areathat has a perimeter defined by the ring. If sample flows from inside tooutside of the ring area, then there is a net flow of the sample acrossfrom within the perimeter of the ring to outside of the perimeter of thering.

In use, a sample may be deposited onto the sample contact area of atleast one of the plates of the device while in the open configuration,closing the plates into the closed configuration and placing the clampin the active mode; and rapidly changing the temperature of the samplein the clamped area, as described above and below.

Size of Clamp Ring: In some embodiments, the clamp ring has an averagelateral size or diameter of 1 mm, 2 mm, 5 mm, 6 mm, 8 mm, 10 mm, 15 mm,20 mm, 30 mm, 40 mm, 50 mm or in a range between any of the two values.In some embodiments, the clamp ring has a preferred average lateral sizeor diameter 5 mm, 6 mm, 8 mm, 10 mm, 15 mm, 20 mm, or in a range betweenany of the two values.

Width of Clamp Ring: In some embodiments, the width of the clamp is 100um, 300 um, 500 um, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 10 mm, 15 mm, or in arange between any of the two values. In some embodiments, the preferredwidth of the clamp is 1 mm, 2 mm, 3 mm, or in a range between any of thetwo values.

Contact Curvature of Clamp Ring: In some embodiments, the radius ofcurvature of one clamp to contact the device is 0.1 mm, 0.2 mm, 0.5 mm,1 mm, 2 mm, 5 mm, 10 mm, 15 mm, 20 mm, or in a range between any of thetwo values. In some embodiments, the preferred radius of curvature ofone clamp to contact the device is 0.2 mm, 0.5 mm, 1 mm, 2 mm, or in arange between any of the two values.

Width Difference of two Clamp Ring: In some embodiments, for one clampset, the width of one clamp ring is larger than the other clamp ring foreasy alignment. In some embodiments, for one clamp set, the width of oneclamp ring is larger than the other clamp ring by 0.2 mm, 0.5 mm, 1 mm,2 mm, 3 mm, 5 mm, 10 mm. 20 mm, or in a range between any of the twovalues. In some preferred embodiments, for one clamp set, the width ofone clamp ring is larger than the other clamp ring by 0.2 mm, 0.5 mm, 1mm, or in a range between any of the two values.

Pressure of Clamp Ring: The device, kit, system, or method of any priorembodiments, wherein the pressure provided by clamp on the card is 5PSI, 10 PSI, 30 PSI, 60 PSI, 90 PSI, 100 PSI, 150 PSI, 200 PSI, 500 PSI1000 pSI, or in average between any of the two values. The device, kit,system, or method of any prior embodiments, wherein the preferredpressure provided by clamp on the card is less than 30 PSI, less than 60PSI, and less than 90 PSI.

B. Quickly Isolating a Portion of a Sample and Assay with RapidTemperature Change

According to the present invention, a sample holder comprised a firstplate and a second plate, where a liquid sample is sandwiched betweenthe plates. In some embodiments, the two plate are fixed to each other.In some embodiments, the two plates are movable relative to each other.In some embodiments, there are spacers between the two plates toregulate the spacing between the two plates.

According to the present invention, a clamp structure comprises a tworings, wherein the clamp has different configuration: inactiveconfiguration and active configuration. In an inactive configuration,the two rings of the claim are open and the two rings do not insert anycompression force on the sample plates. And in an activationconfiguration, the rings are being pushed towards to each other andinsert a compressing force on the areas of a sample holder that areunder the rings, and the comprising pinch force pinches the sampleholder area under the ring together. In some embodiments, the pinchingof the sample holder can reduce the sample in the inside of the clampring to flow to the outside of the clamp.

According the present invention, during a thermal cycling, a clamp isused and active, and the use of the clamp reduces the flow of the liquidsample in the inside of the ring to the outside of the ring. The use ofthe claim reduces the liquid sample flow and hence the energy exchangebetween the sample in the inside of the ring to the outside of the ring,and reduce the heating energy for heating up the sample inside of theclamp, and increase the thermal cycling time.

According the present invention, during a thermal cycling, the use of aclamp can reduce air bubbles during thermal cycling, which in turnimprove thermal cycling quality.

In some embodiments, the method may comprise thermocycling the samplethrough a plurality of cycles that comprise increasing the temperatureof the reaction mix to a temperature of at least 90° C. and thendecreasing the temperature of the reaction mix to one or moretemperatures in the range of 40° C. to 80° C. In a particularembodiment, the method may comprise thermocycling the sample through aplurality of cycles that comprise increasing the temperature of thereaction mix to a temperature of at least 90° C. and then decreasing thetemperature of the reaction mix to one or more temperatures in the rangeof 40° C. to 65° C., then increasing the temperature of the reaction mixto one or more temperatures in the range of 60° C. to 65° C. The samplemay be thermocycled through 10 to 50 cycles, although some protocols canuse more or less cycles.

In particular embodiments, the method may be for performing quantitativereal-time PCR (qPCR). In these embodiments, the method may comprisethermocycling a reaction mix using a the present device, wherein thereaction mix comprises a pair of PCR primers, a polymerase, afluorescence-quencher probe oligonucleotide, dNTPs and a template (aswell as any other necessary components for PCR, e.g., Mg²⁺); and in eachcycle, measuring a fluorescent signal generated by cleavage of a labelfrom the fluorescence-quencher probe oligonucleotide. The fluorescentsignal may be a lump-sum signal or may be done by imaging the sample. Inthese embodiments, the thermocycling may done by activating a heatsource configured to radiate electromagnetic radiation towards a ringarea. Cleavage of the label may be measured by detecting fluorescence ofthe reaction mixture during at least some of the cycles. In someembodiments, the fluorescence-quencher probe oligonucleotide comprises afluorophore and a quencher, and the polymerase cleaves the fluorophoreor quencher from the fluorescence-quencher probe oligonucleotide,thereby generating a fluorescent signal. The fluorescent signal overtime, and the amount of the template in the sample can be estimatedusing the plotted signal.

In some embodiments, a subject biosensor can be used diagnose a pathogeninfection by detecting a target nucleic acid from a pathogen in asample. The target nucleic acid may be, for example, from a virus thatis selected from the group comprising human immunodeficiency virus 1 and2 (HIV-1 and HIV-2), human T-cell leukaemia virus and 2 (HTLV-1 andHTLV-2), respiratory syncytial virus (RSV), adenovirus, hepatitis Bvirus (HBV), hepatitis C virus (HCV), Epstein-Barr virus (EBV), humanpapillomavirus (HPV), varicella zoster virus (VZV), cytomegalovirus(CMV), herpes-simplex virus 1 and 2 (HSV-1 and HSV-2), human herpesvirus8 (HHV-8, also known as Kaposi sarcoma herpesvirus) and flaviviruses,including yellow fever virus, dengue virus, Japanese encephalitis virus,West Nile virus and Ebola virus. The present invention is not, however,limited to the detection of nucleic acid, e.g., DNA or RNA, sequencesfrom the aforementioned viruses, but can be applied without any problemto other pathogens important in veterinary and/or human medicine.

Human papillomaviruses (HPV) are further subdivided on the basis oftheir DNA sequence homology into more than 70 different types. Thesetypes cause different diseases. HPV types 1, 2, 3, 4, 7, 10 and 26-29cause benign warts. HPV types 5, 8, 9, 12, 14, 15, 17 and 19-25 and46-50 cause lesions in patients with a weakened immune system. Types 6,11, 34, 39, 41-44 and 51-55 cause benign acuminate warts on the mucosaeof the genital region and of the respiratory tract. HPV types 16 and 18are of special medical interest, as they cause epithelial dysplasias ofthe genital mucosa and are associated with a high proportion of theinvasive carcinomas of the cervix, vagina, vulva and anal canal.Integration of the DNA of the human papillomavirus is considered to bedecisive in the carcinogenesis of cervical cancer. Humanpapillomaviruses can be detected for example from the DNA sequence oftheir capsid proteins L1 and L2. Accordingly, the method of the presentinvention is especially suitable for the detection of DNA sequences ofHPV types 16 and/or 18 in tissue samples, for assessing the risk ofdevelopment of carcinoma.

Other pathogens that may be detected in a diagnostic sample using thepresent method include, but are not limited to: Varicella zoster,Staphylococcus epidermidis, Escherichia coli, methicillin-resistantStaphylococcus aureus (MSRA), Staphylococcus aureus, Staphylococcushominis, Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcuscapitis, Staphylococcus wameri, Klebsiella pneumoniae, Haemophilusinfluenzae, Staphylococcus simulans, Streptococcus pneumoniae andCandida albicans; gonorrhea (Neisseria gorrhoeae), syphilis (Treponenapallidum), clamydia (Clamyda tracomitis), nongonococcal urethritis(Ureaplasm urealyticum), chancroid (Haemophilus ducrey), trichomoniasis(Trichomonas vaginalis); Pseudomonas aeruginosa, methicillin-resistantStaphlococccus aureus (MSRA), Klebsiella pneumoniae, Haemophilisinfluenzae, Staphylococcus aureus, Stenotrophomonas maltophilia,Haemophilis parainfluenzae, Escherichia coli, Enterococcus faecalis,Serratia marcescens, Haemophilis parahaemolyticus, Enterococcus cloacae,Candida albicans, Moraxiella catarrhalis, Streptococcus pneumoniae,Citrobacter freundii, Enterococcus faecium, Klebsella oxytoca,Pseudomonas fluorscens, Neiseria meningitidis, Streptococcus pyogenes,Pneumocystis Klebsella pneumoniae Legionella pneumophila, Mycoplasmapneumoniae, and Mycobacterium tuberculosis, etc., as well as others.

FIG. 1A shows a sectional view of an embodiment of the system of thepresent invention, comprising a sample holder and a clamping structure,wherein a fluidic sample is sandwiched between the two plates. A clamphas two jaws that can, when the clamp is active, push against each otherto insert a force on a subject between the two jaws. Panel (A)illustrates that when the clamping structure is not activated, the twojaws of the clamp, which have a ring shape, are open, and hence does notassert a force to push the two plates. Panel (B) illustrates that theclamp is activated, where a force is applied by the clamping structureto pinch the sample holder area that is pressed by the clamp. Theactivation of the clamping structure, which clamps the area of thesample holder under the clamp, will reduce or prevent a fluid samplefrom flowing outside the chamber as heat is delivered theheating/cooling layer. In some embodiments, the first plate and thesecond plate are fixed relative to each other. In some embodiments, thefirst plate and the second plate are movable relative to each other.

The term “pinch the sample holder”, “pinch the two plates” or “theplates get pinched” means that when a sample is sandwiched between twoplates wherein at least one of the plates is flexible, and when a ringshaped clamp clamps on the sample holder, the clamp deforms the area ofthe flexible plates that is compressed by the clamp, hence reducing thespacing between the plates in that area. In some cases, the platespacing in the compress area is zero, it is termed “the plates getcompletely pinched”. The pinch of the plate will reduce the sampleliquid flow from inside of the ring to outside.

FIG. 1B shows a top view of an embodiment of one side of a ring clamp.Each ring has a width and a circumference. It shows a circular shapedring clamp and a rectangle shaped ring clamp. As described above, thering clamp may have another shapes.

FIG. 2 . shows a sectional view of an embodiment of the system of thepresent invention, comprising a sample holder, and a clamping structure.The sample holder comprises a first plate and a second plate that aremovable to each other, where in the second plate a well. Panel (A)illustrates the system before the ring clamping structure is activated,where the two rings of the clamp does not assert a force to push the twoplate together. Panel (B) illustrates the system after a force isapplied by the clamping structure. The activation of the clampingstructure will prevent a fluid sample from flowing outside the chamberas heat is delivered the heating/cooling layer.

FIG. 3 . shows a sectional view of an embodiment of the system of thepresent invention, comprising a sample holder and a clamping structure.The sample holder comprises a first plate, a second plate with spacersthat are fixed on the inner surface. Panel (A) illustrates the systembefore the clamping structure is activated. Panel (B) illustrates thesystem after a force is applied by the clamping structure. Theactivation of the clamping structure will prevent a fluid sample fromflowing outside the chamber as heat is delivered the heating/coolinglayer.

FIG. 4 . shows a sectional view of an embodiment of the system of thepresent invention, comprising a first plate, a second plate with a welland spacers that are fixed on the inner surface, and a clampingstructure. Panel (A) illustrates the system before the clampingstructure is activated. Panel (B) illustrates the system after a forceis applied by the clamping structure. The activation of the clampingstructure will prevent a fluid sample from flowing outside the chamberas heat is delivered the heating/cooling layer.

FIG. 5 . shows exemplary embodiments of two types of clampingstructures. Panel (A) comprises a support with a one-spring ringstructure. Panel (B) comprises a support with a four-spring ringstructure. In certain embodiments, one or both of the jaws of the clampare flexible.

In certain embodiments, when a clamp is activated, the two jaws of theclamp are aligned to each other as shown in the image. In certainembodiments, the two jaws of the clamp are misaligned (not on top ofeach other) at least at certain part of the jaws.

In certain embodiments, at least one jaw of the clamp has a sharp edgein contact with the sample holder, such as the round edges shown inFIGS. 1-5 . In certain embodiments, one of the jaws has a sharp edge andthe other jaw has a flow surface, as shown in FIG. 1-5 ; sucharrangement makes an alignment between the two jaws easier.

One embodiment of the present invention is a device for rapidly changingthe temperature of a fluidic sample, comprising: a first plate, a secondplate, and a clamping structure, wherein:

-   -   i. the first plate and the second plate have on their inner        surface a sample contact area for contacting a fluidic sample,        wherein the sample contact area of the first plate and the        second plate face each other, are separated by a separation        distance of 200 um or less, and have an area at least times of        the separation distance, and are capable of sandwiching the        sample between them;    -   ii. the clamping structure comprises a top ring and a bottom        ring that are movable to each other and comprise:        -   a. an open configuration, wherein the top ring and the            bottom ring do not push the first plate and second plate            together; and        -   b. a closed configuration, wherein the top ring and the            bottom ring assert a force again each other to push the            first plate and the second plate in the closed            configuration, deforming an area of the plates that is            compressed by the clamp, leading to a reduction of the            spacing between the two plate in that area, wherein the            reduction of the plate spacing reduces, during thermal            cycling or temperature changing, compared to without using a            clamping structure, reduces the flow of a sample from the            inside to the outside of a ring area.

In another embodiment of the present invention, a device for rapidlychanging the temperature of a fluidic sample, comprises: a first plate,a second plate with a well, and a clamping structure, wherein:

-   -   i. the first plate and the second plate with a well have on        their inner surface a sample contact area for contacting a        fluidic sample; wherein the sample contact area of the first        plate and the second plate face each other, are separated by a        distance of 200 um or less, and are capable of sandwiching the        sample between them;    -   ii. the clamping structure comprises a top ring and a bottom        ring that are movable to each other and comprise:        -   a. an open configuration, wherein the top ring and the            bottom ring do not push the first plate and the second plate            with a well together; and        -   b. a closed configuration, wherein the top ring and the            bottom ring assert a force again each other to push the            first plate and the second plate with a well in the closed            configuration, so the flow of a sample from the inside to            the outside of a ring area during thermal cycling is reduced            compared to without using a clamping structure.

As illustrated in FIG. 1A (Panel A and Panel B) and FIG. 2 (Panel A andPanel B), the sectional views of the embodiments show an openconfiguration (before clamp activation) and a closed configuration(after the clamp is activated). In FIG. 1A, the first plate and secondplate are flat. In FIG. 2 , the second plate comprises a well.

In another embodiment of the present invention, a device for rapidlychanging the temperature of a fluidic sample, comprises: a first plate,a second plate, spacers, and a clamping structure, wherein:

-   -   i. the first plate and/or second plate comprise spacers fixed to        the inner surface of the first plate and/or second plate,    -   ii. the first plate and the second plate comprise on their inner        surface a sample contact area for contacting a fluidic sample;        wherein the sample contact area of the first plate and the        second plate face each other, are separated by a distance of 200        um or less, and are capable of sandwiching the sample between        them,    -   iii. the clamping structure comprises a top ring and a bottom        ring that are movable to each other and comprise:        -   a. an open configuration, wherein the top ring and the            bottom ring do not push the first plate and the second plate            with a well together; and        -   b. a closed configuration, wherein the top ring and the            bottom ring assert a force again each other to push the            first plate and the second plate with a well in the closed            configuration, so the flow of a sample from the inside to            the outside of a ring area during thermal cycling is reduced            compared to without using a clamping structure.

In yet another embodiment of the present invention, a device for rapidlychanging the temperature of a fluidic sample, comprises: a first plate,a second plate with a well, spacers, and a clamping structure, wherein:

-   -   i. the first plate and/or second plate with a well comprise        spacers fixed to the inner surface of the first plate and/or        second plate,    -   ii. the first plate and the second plate with a well comprise on        their inner surface a sample contact area for contacting a        fluidic sample; wherein the sample contact area of the first        plate and the second plate face each other, are separated by a        distance of 200 um or less, and are capable of sandwiching the        sample between them,    -   iii. the clamping structure comprises a top ring and a bottom        ring that are movable to each other and comprise:        -   a. an open configuration, wherein the top ring and the            bottom ring do not push the first plate and the second plate            with a well together; and        -   b. a closed configuration, wherein the top ring and the            bottom ring assert a force again each other to push the            first plate and the second plate with a well in the closed            configuration, so the flow of a sample from the inside to            the outside of a ring area during thermal cycling is reduced            compared to without using a clamping structure.

As illustrated in FIG. 3 (Panel A and Panel B) and FIG. 4 (Panel A andPanel B), the sectional views of the embodiments show an openconfiguration (before clamp activation) and a closed configuration(after the clamp is activated) wherein spacers are positioned betweenthe first plate and the second plate to regulate the distance betweenthe two plates (i.e., the spacing of the first plate and the secondplate), to regulate the sample thickness. The spacers allow thethickness of the sample between the first plate and the second plate tobe uniform over a large area, even when the first plate and second plateare thin and flexible. In FIG. 3 , the first plate and the second plateare flat. In FIG. 4 , the second plate comprises a well.

In certain embodiments, the pressure inserted by clamp is configured tocompletely pinched the flexible plate area that is clamped by the ringclamp. A complete pinch of the plates stops fluidic communicationbetween the liquid sample inside of the ring clamp and outside.

In certain embodiments, the pressure inserted by clamp is configured tocrush the spacers to pinch the flexible plate area. A spacer can breakinto piece when the deformation of a spacer is beyond its elasticdeformation limit (metals are an exception of this).

A device for rapidly changing the temperature of a fluidic sample,comprising: a first plate, a second plate, and a clamping structure,wherein:

-   -   i. the first plate and the second plate have on their inner        surface a sample contact area for contacting a fluidic sample;        wherein the sample contact area of the first plate and the        second plate face each other, are separated by a distance of 200        um or less, and are capable of sandwiching the sample between        them;    -   ii. the clamping structure comprises a top ring and a bottom        ring that are movable to each other and comprise:        -   a. an open configuration, wherein the top ring and the            bottom ring do not push the first plate and second plate            together; and        -   b. a closed configuration, wherein the top ring and the            bottom ring assert a force again each other to push the            first plate and the second plate in the closed            configuration, so the flow of a sample from the inside to            the outside of a ring area during thermal cycling is reduced            compared to without using a clamping structure.

A device for rapidly changing the temperature of a fluidic sample,comprising: a first plate, a second plate with a well, and a clampingstructure, wherein:

-   -   i. the first plate and the second plate with a well have on        their inner surface a sample contact area for contacting a        fluidic sample; wherein the sample contact area of the first        plate and the second plate face each other, are separated by a        distance of 200 um or less, and are capable of sandwiching the        sample between them;    -   ii. the clamping structure comprises a top ring and a bottom        ring that are movable to each other and comprise:        -   a. an open configuration, wherein the top ring and the            bottom ring do not push the first plate and the second plate            with a well together; and        -   b. a closed configuration, wherein the top ring and the            bottom ring assert a force again each other to push the            first plate and the second plate with a well in the closed            configuration, so the flow of a sample from the inside to            the outside of a ring area during thermal cycling is reduced            compared to without using a clamping structure.

A device for rapidly changing the temperature of a fluidic sample,comprising: a first plate, a second plate, spacers, and a clampingstructure, wherein:

-   -   i. the first plate and/or second plate comprise spacers fixed to        the inner surface of the first plate and/or second plate,    -   ii. the first plate and the second plate comprise on their inner        surface a sample contact area for contacting a fluidic sample;        wherein the sample contact area of the first plate and the        second plate face each other, are separated by a distance of 200        um or less, and are capable of sandwiching the sample between        them,    -   iii. the clamping structure comprises a top ring and a bottom        ring that are movable to each other and comprise:        -   a. an open configuration, wherein the top ring and the            bottom ring do not push the first plate and the second plate            with a well together; and        -   b. a closed configuration, wherein the top ring and the            bottom ring assert a force again each other to push the            first plate and the second plate with a well in the closed            configuration, so the flow of a sample from the inside to            the outside of a ring area during thermal cycling is reduced            compared to without using a clamping structure.

A device for rapidly changing the temperature of a fluidic sample,comprising: a first plate, a second plate with a well, spacers, and aclamping structure, wherein:

-   -   i. the first plate and/or second plate with a well comprise        spacers fixed to the inner surface of the first plate and/or        second plate,    -   ii. the first plate and the second plate with a well comprise on        their inner surface a sample contact area for contacting a        fluidic sample; wherein the sample contact area of the first        plate and the second plate face each other, are separated by a        distance of 200 um or less, and are capable of sandwiching the        sample between them,    -   iii. the clamping structure comprises a top ring and a bottom        ring that are movable to each other and comprise:        -   a. an open configuration, wherein the top ring and the            bottom ring do not push the first plate and the second plate            with a well together; and        -   b. a closed configuration, wherein the top ring and the            bottom ring assert a force again each other to push the            first plate and the second plate with a well in the closed            configuration, so the flow of a sample from the inside to            the outside of a ring area during thermal cycling is reduced            compared to without using a clamping structure.

A method for rapidly changing the temperature of a fluidic sample,comprising: a first plate, a second plate, and a clamping structure,wherein:

-   -   i. the first plate and the second plate have on their inner        surface a sample contact area for contacting a fluidic sample;        wherein the sample contact area of the first plate and the        second plate face each other, are separated by a distance of 200        um or less, and are capable of sandwiching the sample between        them;    -   ii. the clamping structure comprises a top ring and a bottom        ring that are movable to each other and comprise:        -   a. an open configuration, wherein the top ring and the            bottom ring do not push the first plate and second plate            together; and        -   b. a closed configuration, wherein the top ring and the            bottom ring assert a force again each other to push the            first plate and the second plate in the closed            configuration, so the flow of a sample from the inside to            the outside of a ring area during thermal cycling is reduced            compared to without using a clamping structure.

A method for rapidly changing the temperature of a fluidic sample,comprising: a first plate, a second plate with a well, and a clampingstructure, wherein:

-   -   iii. the first plate and the second plate with a well have on        their inner surface a sample contact area for contacting a        fluidic sample; wherein the sample contact area of the first        plate and the second plate face each other, are separated by a        distance of 200 um or less, and are capable of sandwiching the        sample between them;    -   iv. the clamping structure comprises a top ring and a bottom        ring that are movable to each other and comprise:        -   c. an open configuration, wherein the top ring and the            bottom ring do not push the first plate and the second plate            with a well together; and            -   a closed configuration, wherein the top ring and the                bottom ring assert a force again each other to push the                first plate and the second plate with a well in the                closed configuration, so the flow of a sample from the                inside to the outside of a ring area during thermal                cycling is reduced compared to without using a clamping                structure.

A method for rapidly changing the temperature of a fluidic sample,comprising: a first plate, a second plate, spacers, and a clampingstructure, wherein:

-   -   i. the first plate and/or second plate comprise spacers fixed to        the inner surface of the first plate and/or second plate,    -   ii. the first plate and the second plate comprise on their inner        surface a sample contact area for contacting a fluidic sample;        wherein the sample contact area of the first plate and the        second plate face each other, are separated by a distance of 200        um or less, and are capable of sandwiching the sample between        them,    -   iii. the clamping structure comprises a top ring and a bottom        ring that are movable to each other and comprise:        -   a. an open configuration, wherein the top ring and the            bottom ring do not push the first plate and the second plate            with a well together; and        -   b. a closed configuration, wherein the top ring and the            bottom ring assert a force again each other to push the            first plate and the second plate with a well in the closed            configuration, so the flow of a sample from the inside to            the outside of a ring area during thermal cycling is reduced            compared to without using a clamping structure.

A method for rapidly changing the temperature of a fluidic sample,comprising: a first plate, a second plate with a well, spacers, and aclamping structure, wherein:

-   -   i. the first plate and/or second plate with a well comprise        spacers fixed to the inner surface of the first plate and/or        second plate,    -   ii. the first plate and the second plate with a well comprise on        their inner surface a sample contact area for contacting a        fluidic sample; wherein the sample contact area of the first        plate and the second plate face each other, are separated by a        distance of 200 um or less, and are capable of sandwiching the        sample between them,    -   iii. the clamping structure comprises a top ring and a bottom        ring that are movable to each other and comprise:        -   a. an open configuration, wherein the top ring and the            bottom ring do not push the first plate and the second plate            with a well together; and        -   b. a closed configuration, wherein the top ring and the            bottom ring assert a force again each other to push the            first plate and the second plate with a well in the closed            configuration, so the flow of a sample from the inside to            the outside of a ring area during thermal cycling is reduced            compared to without using a clamping structure.

The device or method of any prior embodiments further comprising aheating layer. The device or method of any prior embodiments, whereinthe heating layer is positioned on the inner surface, the outer surface,or inside of one of the plates.

The device or method of any prior embodiments, wherein the heating layeris configured to heat a relevant volume of the sample, wherein therelevant volume of the sample is a portion or an entirety of the samplethat is being heated to a desired temperature.

The device or method of any prior embodiments further comprising acooling layer. The device or method of any prior embodiments, whereinthe cooling layer is positioned on the inner surface, the outer surface,or inside one of the plates. The device or method of any priorembodiments wherein the cooling layer is configured to cool the relevantsample volume.

The device or method of any prior embodiments, wherein the cooling layercomprises a layer of material that has a thermal conductivity to thermalcapacity ratio of 0.6 cm²/sec or larger.

The device or method of any prior embodiments, wherein the clampingstructure is attached to either one or both of the first and secondplates, and wherein the clamping structure is configured to hold thedevice and regulate the thickness of the sample layer during the heatingof the device.

The device, kit, system, or method of any prior embodiments, wherein thethickness of the clamp is 100 um, 300 um, 500 um, 1 mm, 2 mm, 3 mm, 4mm, 5 mm, 10 mm, 15 mm, 20 mm, 30 mm or in a range between any of thetwo values.

The device, kit, system, or method of any prior embodiments, wherein thepreferred thickness of the clamp is 1 mm, 2 mm, 10 mm, and 15 mm.

The device, kit, system, or method of any prior embodiments, wherein thecircumference of the clamp is 5 mm, 10 mm, 20 mm, 30 mm, 38 mm, 50 mm,62 mm, 100 mm or in a range between any of the two values.

The device, kit, system, or method of any prior embodiments, wherein thepreferred circumference of the clamp is 38 mm and 62 mm.

The device, kit, system, or method of any prior embodiments, wherein thematerial of clamp includes, but not limited to, glass, quartz, oxides,silicon-dioxide, silicon-nitride, hafnium oxide (HfO), aluminum oxide(AlO), semiconductors: (silicon, GaAs, GaN, etc.).

The device, kit, system, or method of any prior embodiments, wherein thematerial of clamp includes, but not limited to metals (e.g. gold,silver, coper, aluminum, Ti, Ni, etc.), ceramics, or any combinations ofthereof.

The device, kit, system, or method of any prior embodiments, wherein thematerial of clamp includes, but not limited to, polymers (e.g. plastics)or amorphous organic materials. The polymer materials include, but notlimited to, acrylate polymers, vinyl polymers, olefin polymers,cellulosic polymers, noncellulosic polymers, polyester polymers, Nylon,cyclic olefin copolymer (COC), poly(methyl methacrylate) (PMMA),polycarbonate (PC), cyclic olefin polymer (COP), liquid crystallinepolymer (LCP), polyimide (PA), polyethylene (PE), polyimide (PI),polypropylene (PP), poly(phenylene ether) (PPE), polystyrene (PS),polyoxymethylene (POM), polyether ether ketone (PEEK), polyether sulfone(PES), poly(ethylene phthalate) (PET), polytetrafluoroethylene (PTFE),polyvinyl chloride (PVC), polyvinylidene fluoride (PVDF), polybutyleneterephthalate (PBT), fluorinated ethylene propylene (FEP),perfluoroalkoxyalkane (PFA), polydimethylsiloxane (PDMS), rubbers, orany combinations of thereof.

The device, kit, system, or method of any prior embodiments, wherein thematerial of clamp includes, but not limited to glass, quartz, oxides,silicon-dioxide, silicon-nitride, hafnium oxide (HfO), aluminum oxide(AlO), semiconductors: (silicon, GaAs, GaN, etc.), plastics, metals(e.g. gold, silver, coper, aluminum, Ti, Ni, etc.), ceramics, or anycombinations of thereof.

C. Fast Temperature Changes

Working Principle

One aspect of the present invention is to reduce thermal cycling time,to reduce the heating energy used for such cycling, to increase energyefficiency, and to reduce total power consumption.

The thermal cycling time (speed), heating energy, energy efficiency, andpower consumption are related. When more heating energy is needed inraising the temperature of a given sample, the more energy must beremoved in cooling the sample, which, in turn, needs more time and/ormore energy to perform the cooling.

Many thermal cyclers in prior art require a use of a significant amountof heating energy to the sample holder (e.g. plastic chamber walls)rather than to the sample; a use of lateral thermal conduction throughlarge thermal mass and poor-thermal conduction materials of the sampleholder as the major cooling channel to cool the sample (note that amaterial needs to absorb and release energy to perform a thermalconduction); a use of conductive cooling as major cooling method, and/ora use of an extra cooling gas or a moving cooling block. Theseapproaches lead to issues of long thermal cycling time, high heatingenergy, low energy efficiency, bulky apparatus, and/or high cost.

Based on theoretical and experimental investigations, the presentinvention provides solutions to certain drawbacks in a sample thermalcycling in the prior arts.

To illustrate the working principle of the present invention, let uslook at the energy components in heating and cooling a sample by athermal cycler. The heating and cooling share three energy components:(i) one related to thermal mass (i.e. a material's ability to absorb andstore energy; larger the thermal mass, more energy needed to be addedfor heating up and more energy needed to be removed in cooling), (ii)heat loss by thermal radiation, and (iii) heat loss by thermalconduction/convection. To heat fast, all three energy components need tobe small. But to cool fast, the first energy component needs to besmall, but at least one of the last two energy components needs to belarge.

Through theoretical and experimental investigation, the presentinvention balances and/or optimizes the three energy components forachieving rapid heating and cooling. Particularly, in certainembodiments, the present invention reduces the thermal mass that must beheated in a thermal cycle, limits lateral thermal conduction, and usesradiative heat loss as a primary way to remove energy from the heatedsample.

According to the present invention, the cooling of a sample issignificantly by thermal radiative cooling, not by thermal conductioncooling. Therefore, in a thermal cycling, most or a significant part ofthe non-sample materials on a sample holder do not absorb and release asmuch energy as that in a thermal conduction dominated system.

One aspect of the present invention provides devices and methods thatreduce the heating to non-sample materials on the sample holder.

Another aspect of the present invention provides devices and methodsthat reduce lateral thermal conduction through large thermal mass andpoor-thermal conduction materials on the sample holder.

Another aspect of the present invention provides devices and methodsthat use thermal radiative cooling as the major cooling channel to coolthe sample.

Another aspect of the present invention provides devices and methodsthat place spacers between to plates (i.e. walls) that sandwich asample. The spacers provides good sample uniformity over a large area,even when the plates are thin (e.g. 25 um thick) and flexible. Withoutspacers, it can be difficult to achieve a uniform sample thickness, whenthe two plates that confine the sample become very thin.

Another aspect of the present invention provides devices and methodsthat make the device operation easier.

According to the present invention, the thermal radiative cooling uses amaterial layer are configured (in terms of materials and shape) that hasgood thermal radiative cool properties during the cooling, and a lowthermal mass (hence a low heating energy) during heating.

According to the present invention, the sample holder is configured tolimit/minimize the thermal conduction cooling.

According to the present invention, the sample thickness, the firstplate and the second plate (which are facing each other) of the samplechamber wall thickness are configured to reduce the lateral thermalconduction (i.e. in the direction of the plate).

According to the present invention, in some embodiments, the thermalradiative cooling layer is the same heating/cooling layer of the heatinglayer, but the ratio of the cooling zone to the heating zone, thematerial properties, and the material thickness and geometry areconfigured to make the heating/cooling layer has a low thermal mass inheating and high rate of thermal radiative cooling.

Another objective of the present invention is to make one cycle of asample temperature change (e.g. from 95° C. to 55° C.) in a few secondsor even sub-second (e.g., 0.7 second).

Another aspect of the present invention is that it provides usefuldevices and methods for isothermal nucleic acid amplification, where asample temperature needs to be raised from environment to an elevatedtemperature (i.e. 65° C.) and keep there for a period of time (i.e.,5-10 min). One aspect of the present invention is to raise thetemperature fast, to use less energy, and to make the apparatus compact,lightweight, and portable.

One aspect of the present invention is that the thermal masses of thecard as well as the sample are minimized to reduce the energy needed forheating and the energy to be removed for cooling.

Another aspect of the present invention is that in certain embodiments,only a small portion of the sample is heated and/or cooled.

Another aspect of the present invention is that it uses a thin highthermal conductivity layer that has an area size larger than that of therelevant sample area.

Another aspect of the present invention is that it uses a thin highthermal conductivity layer that has an area size larger than the heatingzone area.

Another aspect of the present invention provides devices and methodsthat reduce the heating to non-sample materials on the sample holder.

Another aspect of the present invention provides devices and methodsthat reduce lateral thermal conduction in large thermal mass andpoor-thermal conduction materials on the sample holder.

Another aspect of the present invention provides devices and methodsthat use thermal radiative cooling as the major cooling channel to coolthe sample.

Another aspect of the present invention is that it can achieve fastthermal cycling without using a cooling gas.

Another aspect of the present invention is that the thermal masses ofthe card as well as the sample are minimized to reduce the energy neededfor heating and the energy to be removed for cooling.

Another aspect of the present invention is that the radiative coolingand convention cooling are adjusted for rapid cooling.

Another aspect of the present invention is that heat sink for radiativecooling and/or convention cooling is used for rapid cooling.

One embodiment of a sample thermal cycling apparatus in the presentinvention (as illustrated in FIG. 6 ) comprises: (i) a sample holder,termed “RHC (rapid heating and cooling) Card” or “sample card”, thatallows a rapid heating and cooling of a sample on the card; (ii) aheating source, (iii) an extra heat sink (optional), (iv) a temperaturecontrol system, and (v) a signal monitoring system (optional). Thetemperature control system and signal monitoring system are notexplicitly illustrated in FIG. 6 , but may be used to control the outputof the heating source. In some embodiments, a signal sensor is includedto detect optical signals from samples on the sample holder. Note thatcertain embodiments of the present invention can have just one orseveral components illustrated in FIG. 6 .

FIGS. 2A and 2B show sectional views of two embodiments of the device ofthe present invention. FIG. 7A shows an embodiment comprising a separateheating layer (112-1) and a separate cooling layer (112-2), wherein theheating layer (112-1) is on the outer surface of one of the plates andthe cooling layer (112-2) is on the outer surface of the other plate.FIG. 7B shows an embodiment comprising a heating layer (112-1) and acooling layer (112-2), wherein the heating layer (112-1) and the coolinglayer (112-2) are structurally distinct but in contact with each other,and the two layers are both on the outer surface of one of the plates.

SH-1 One detailed description of one embodiment of a RHC card in thepresent invention is that a device for rapidly changing the temperatureof a fluidic sample, comprising:

a first plate (10), a second plate (20), a heating layer (112-1), and acooling layer (112-2), wherein:

each of the first plate and the second plate has, on its respectiveinner surface, a sample contact area for contacting a fluidic sample;wherein the sample contact areas face each other, are separated by anaverage separation distance of 200 um or less, and are capable ofsandwiching the sample between them;

the heating layer is:

positioned on the inner surface, the outer surface, or inside of one ofthe plates, and

configured to heat a relevant volume of the sample, wherein the relevantvolume of the sample is a portion or an entirety of the sample that isbeing heated to a desired temperature; and

the cooling layer is:

positioned on the inner surface, the outer surface, or inside of one ofthe plates, and

configured to cool the relevant sample volume; and

comprises a layer of material that that has a thermal conductivity tothermal capacity ratio of 0.6 cm²/sec or larger;

wherein the distance between the cooling layer and a surface of therelevant sample volume is zero or less than a distance that isconfigured to make the thermal conductance per unit area between thecooling layer and the surface of the relevant sample volume equal to 70W/(m²·K) or larger; and

wherein, in some embodiments, the heating layer and cooling layer arethe same material layer that have a heating zone and cooling zone, andwherein the heating zone and cooling zone can have the same area ordifferent areas.

SH-2 Another detailed description of one embodiment of a RHC card(sample holder) in the present invention is that a device for rapidlychanging the temperature of a fluidic sample, comprising:

A first plate (10), a second plate (20), a heating layer (112-1), and acooling layer (112-2), wherein:

each of the first plate and the second plate has, on its respectiveinner surface, a sample contact area for contacting a fluidic sample;wherein the sample contact areas face each other, are separated by anaverage separation distance of 200 um or less, and are capable ofsandwiching the sample between them;

the heating layer is:

positioned on the inner surface, the outer surface, or inside of one ofthe plates, and configured to heat a relevant volume of the sample,wherein the relevant volume of the sample is a portion or an entirety ofthe sample that is being heated to a desired

temperature; and

the cooling layer is:

positioned on the inner surface, the outer surface, or inside of one ofthe plates; and

configured to cool the relevant sample volume; and

comprises a layer of material that that has a thermal conductivity tothermal capacity ratio of 0.6 cm²/sec or larger, wherein the highthermal conductivity to thermal capacity ratio layer has an area largerthan the lateral area of the sample volume;

wherein the distance between the cooling layer and a surface of therelevant sample volume is zero or less than a distance that isconfigured to make the thermal conductance per unit area between thecooling layer and the surface of the relevant sample volume equal to 150W/(m²·K) or larger; and

wherein, in some embodiments, the heating layer and cooling layer arethe same material layer that have a heating zone and cooling zone, andwherein the heating zone and cooling can have the same area or differentareas.

As illustrated in FIGS. 8A and 8B, in some embodiments of the presentinvention, the heating layer and the cooling layer are combined into onelayer (heating/cooling layer) creating a heating zone and cooling zone,where the cooling zone is larger than the heating zone. A sample card100 (also termed “RHC card”) may include two thin plates (10, 20) thatsandwich a fluidic sample (90) between them and a heating/cooling layer(112) is under the sample, and the heating/cooling layer (112) is heatedby a heat source positioned away from the card. According to anembodiment, at the edge of the sample, there are no walls to contain thesample, but the edge of the sample will not flow due to capillary forcesthat keep the shape of the fluidic sample edges.

As illustrated in FIG. 9A, plates 10 and 20 may have inner surfaces 11and 21 that are separated by a spacing 102, according to an embodiment.Spacing 102 may be large when the device is ready to receive a sample(e.g., in an open position). FIG. 9B illustrates a closed configurationof device 100 where spacing 102 is made small (e.g., less than about 200μm) to sandwich a sample 90 between plates 10 and 20. In thisembodiment, heating/cooling layer 112 is positioned on an outer surface22 of plate 20.

SH-3 Another detailed description of one embodiment of a RHC card in thepresent invention is that a device for rapidly changing the temperatureof a fluidic sample, comprising:

a first plate (10), a second plate (20), and a heating/cooling layer(112), wherein:

the first plate (10) and the second plate (20) face each other, and areseparated by a distance from each other;

each of the plates has, on its respective inner surface (11, 21), asample contact area for contacting a fluidic sample; wherein the samplecontact areas are facing each other, are in contact with the sample,confines a sample between them, and have an average separation distance(102) from each other, and the sample;

the heating/cooling layer (112) is on the outer surface (22) of thesecond plate (20); and

the heating/cooling layer is configured to comprise a heating zone and acooling zone; wherein the heat zone is configured to heat the fluidicsample, the cooling zone is configured to cool the sample by thermalradiative cooling;

wherein the heating zone is configured to receive heating energy from aheating source and configured to have an area smaller than the totalarea of the heating/cooling layer; and

wherein at least a part of a heating zone of the heating layer overlapswith the sample area.

SH-4 A device for rapidly changing the temperature of a fluidic sample,comprising:

a first plate (10), a second plate (20), a heating layer (112-1), and acooling layer (112-2), wherein:

the first and second plates are movable relative to each other intodifferent configurations;

each of the first plate and the second plate has, on its respectiveinner surface, a sample contact area for contacting a fluidic sample;wherein the sample contact areas face each other, are separated by anaverage separation distance of 200 um or less, and are capable ofsandwiching the sample between them;

the heating layer:

is positioned on the inner surface, the outer surface, or inside of oneof the plates,

is configured to heat a relevant volume of the sample, wherein therelevant volume of the sample is a portion or an entirety of the samplethat is being heated to a desired temperature; and

the cooling layer is:

positioned on the inner surface, the outer surface, or inside of one ofthe plates; and

configured to cool the relevant sample volume; and

comprises a layer of material that that has a thermal conductivity tothermal capacity ratio of 0.6 cm²/sec or larger;

wherein one of the configurations is an open configuration, in which:the two plates are partially or completely separated apart and theaverage spacing between the plates is at least 300 um;

wherein another of the configurations is a closed configuration which isconfigured after the fluidic sample is deposited on one or both of thesample contact areas in the open configuration; and in the closedconfiguration: at least part of the sample is confined by the two platesinto a layer, wherein the average sample thickness is 200 um or less.

SH-5 A device for rapidly changing the temperature of a fluidic sample,comprising:

a first plate (10), a second plate (20), spacers, a heating layer(112-1), and a cooling layer (112-2), wherein:

the first and second plates are movable relative to each other intodifferent configurations;

each of the first plate and the second plate has, on its respectiveinner surface, a sample contact area for contacting a fluidic sample;wherein the sample contact areas face each other, are separated by anaverage separation distance of 200 um or less, and are capable ofsandwiching the sample between them;

one or both of the plates comprise the spacers and the spacers are fixedon the inner surface of a respective plate;

the spacers have a predetermined substantially uniform height that isequal to or less than 200 microns, and the inter-spacer-distance ispredetermined;

the heating layer is:

positioned on the inner surface, the outer surface, or inside of one ofthe plates, and

configured to heat a relevant volume of the sample, wherein the relevantvolume of the sample is a portion or an entirety of the sample that isbeing heated to a desired temperature; and

the cooling layer is:

positioned on the inner surface, the outer surface, or inside of one ofthe plates; and

configured to cool the relevant sample volume; and

comprises a layer of material that that has a thermal conductivity tothermal capacity ratio of 0.6 cm²/sec or larger;

wherein one of the configurations is an open configuration, in which:the two plates are partially or completely separated apart, the spacingbetween the plates is not regulated by the spacers, and the sample isdeposited on one or both of the plates; and

wherein another of the configurations is a closed configuration which isconfigured after the sample is deposited in the open configuration; andin the closed configuration: at least part of the sample is compressedby the two plates into a layer of highly uniform thickness, wherein theuniform thickness of the layer is confined by the sample contactsurfaces of the plates and is regulated by the plates and the spacers.

In some embodiments, the heating/cooling layer (112) can be on the innersurface (21) or inside the second plate (20), rather than on the outersurface (22) of the second plate (20).

In some embodiments of all embodiments of devices, the RHC card furthercomprises spacers that are positioned between the first and second plateto regulate the distance between the two plates (i.e. the spacing of theplates), and hence to regulate the sample thickness. The spacers canallow the thickness of the sample between the two plates uniform over alarge area, even when the plates are thin and flexible.

In some embodiments, there are more than one heating/cooling layer.

A. Small Relevant Sample Volume (RE Ratio)

Reduction of the sample volume that should be heated or cooled to adesirable temperature can shorten the heating time and cooling time in athermal cycle as well as heating power. A reduction of the sample volumethat will be thermal cycled can be achieved by (a) reducing the entiresample volume or (b) heating just a portion of the sample on the sampleholder. The term “relevant sample” or “relevant sample volume” refers tothe volume of the sample that is being heated and/or cooled to desiredtemperatures during a thermal cycling, and the relevant sample can be aportion or an entire volume of a sample on a sample holder, and there isno fluidic separation between the portion of the sample to the rest ofthe sample.

In some embodiments, the relevant volume of the sample is 0.001 ul,0.005 ul, 0.01 ul, 0.02 ul, 0.05 ul, 0.1 ul, 0.2 ul, 0.5 ul, 1 ul, 2 ul,5 ul, 10 ul, 20 ul, 30 uL, 50 ul, 100 ul, 200 ul, 500 ul, 1 ml, 2 ml, 5ml, or in a range between any of the two values.

In some preferred embodiments, the relevant sample volume is in a rangeof 0.001 uL to 0.1 uL, 0.1 um to 2 uL, 2 uL to 10 uL, 10 uL to 30 uL, 30uL to 100 uL, 100 uL to 200 uL, or 200 uL to 1 mL.

In some preferred embodiments, the relevant sample volume is in a rangeof 0.001 uL to 0.1 uL, 0.1 um to 1 uL, 0.1 uL to 5 uL, or 0.1 uL to 10uL.

In certain embodiments, the ratio of the relevant sample to entiresample volume (RE ratio) is 0.01%, 0.05%, 0.1%, 0.5%, 0.1%, 0.5%, 1%,5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or in a rangebetween any of the two values.

In some preferred embodiments, the RE ratio is in a range of between0.01% and 0.1%, 0.1% and 1%, 1% and 10%, 10% and 30%, 30% and 60%, 60%and 90%, or 90% and 100%.

To heat only a portion of the sample, in some embodiments, the area ofthe heating zone is only a fraction of the sample lateral area, and thefraction (i.e. the ratio of the heating zone to the sample lateral area)is 0.01%, 0.05%, 0.1%, 0.5%, 0.1%, 0.5%, 1%, 5%, 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 99%, or in a range between any of the twovalues.

In some preferred embodiments, the ratio of the heating zone area to thesample lateral area is in a range of between 0.01% and 0.1%, 0.1% and1%, 1% and 10%, 10% and 30%, 30% and 60%, 60% and 90%, or 90% and 99%.

B. Local Heating, High Vertical to Lateral Heat Transfer

When a high-K (high thermal conductivity) layer is (e.g. a metal layer)on the inner surface, the outer surface, or inside of one of the platesof a sample holder (RHC card), to make only a part of the high-K layerand a part of sample volume above the part of the high-K layer to beheated to desired temperatures, while keeping the rest of the high-Klayer and the rest of the sample volume at much lower temperaturesduring a thermal cycling, several conditions must be met. The keyconditions are (1) the heat source must directly heat a portion of thehigh-K layer (the portion is termed “heat zone” e.g., only the portionis directly heated by a LED light or has a local electric heater, whilethe rest is not), (2) the vertical heating transfer between the heatzone and a portion of the sample should be much larger than the lateralheat transfer within the high-K material (i.e. in the lateral directionof the high-K material), (3) the relevant sample should have a largelateral to vertical size ratio, and (4) the heating power of the heatzone must sufficient to heat up the relevant sample volume in a timeframe that lateral heat transfer (i.e., heat conduction) is relativelynegligible.

To satisfy the condition (2) above, the scaled thermal conduction ratio(STC ratio) of the vertical heat transfer from the high-K heating zoneto the sample through the middle layer that is between the high-K andthe sample to the lateral heat transfer inside the high-K layer isdefined as:

${{STC}{ratio}} = {\eta = {0{\text{.025} \cdot \frac{K_{m}K_{s}D^{2}}{{K_{k}\left( {{K_{m}t_{s}} + {K_{s}t_{m}}} \right)}t_{k}}}}}$

wherein K_(k), K_(s), and K_(m) is, respectively, the thermalconductivity of the high-K layer, the relevant sample, and the middlelayer (i.e. the layer between the high-K and the sample), t_(k), t_(s),and t_(m) is, respectively, the thickness of the high-K layer, thesample, and the middle layer; D is the average lateral dimension of therelevant sample, and 0.025 is a scaling factor.

To locally heat a part of the high-K layer and a part of sample volumeabove the part of the high-K layer to desired temperatures, whilekeeping the rest of the high-K layer and the rest of the sample volumeat much lower temperatures during a thermal cycling. In someembodiments, the scaled thermal conduction ratio (STM ratio) is 2 orlarger, 5 or larger, 10 or larger, 20 or larger, 30 or larger, 40 orlarger, 50 or larger, 100 or larger, 1000 or larger, 10000 or larger,10000 or larger, or in a range between any of the two values.

In some preferred embodiments, the scaled thermal conduction ratio (STMratio) is in a range of between 10 to 20, 30 to 50, 100 to 1,000, 1,000to 10,000, or 10,000 to 1,000,000.

To satisfying the condition (2) and (3) above, in some embodiments, thelateral to vertical size (LVS) ratio for relevant sample is 5, 10, 20,50, 70, 100, 200, 300, 400, 500, 600, 700, 800, 800, 1,000, 2,000,5,000, 10,000, 100,000, or in a range between any of the two values.

In some preferred embodiments, the LVS ratio for relevant sample is in arange of 5 to 10, 10 to 50, 50 to 100, 100 to 500, 500 to 1,000, 1,000,to 10,000, or 10,000 to 100,000,

In certain embodiments, the thickness of the relevant sample is reduced(which also can help sample heating speed), and the relevant sample hasa thickness of 0.05 um, 0.1 um, 0.2 um, 0.5 um, 1 um, 2 um, 5 um, 10 um,20 um, 30 um, 40 um, 50 um, 60 um, 70 um, 80 um, 90 um, 100 um, 200 um,300 um, or in a range between any of the two values.

In some preferred embodiments, the relevant sample has a thickness in arange between 0.05 um and 0.5 um, 0.5 um and 1 um, 1 um and 5 um, 5 umand 10 um, 10 um and 30 um, 30 um and 50 um, 50 um and 70 um, 70 um and100 um, 100 um and 200 um, or 200 um and 300 um.

C. Large Sample to Non-Sample Thermal Mass Ratio (NSTM Ratio)

An increase of the sample-to-non-sample thermal mass ratio can shortenheating time, reduce heating energy, and increase energy efficiency. Inan embodiment where a sample is sandwiched between the two plates, athermal mass ratio can be estimated by only considering the relevantsample volume and the portions of the two plates that sandwich therelevant sample, assuming there are no thermal losses in these volumes.Therefore, one parameter to measure a thermal mass ratio is the ratio of“specific area thermal mass” of the relevant sample to the non-sample(the portions of the plates that sandwich the relevant sample as well asthe part heating/cooling layer on the plate portion). The term “specificarea thermal mass” of a material refers to as the volume specific heatof the material multiplying its thickness.

The sample to non-sample thermal mass ratio is a ratio of the usefulheat energy (which directly heat the relevant sample) to the “wastedheat energy (that heats non-sample materials), assuming that the heatlosses by thermal conduction and radiation are negligible.

For examples, water has a volume specific heat of 4.2 J/(cm³·° C.), thusthe area specific heat for a 30 um thick water layer is 1.26×10⁻²J/(cm³·° C.). A PMMA has a volume specific heat of 1.77 J/(cm³·° C.),thus the area specific heat for a 25 um thick PMMA layer is 4.43×10⁻³J/(cm³·° C.), which is ˜2.8 times less than that of 30 um water layer.Gold has a volume specific heat of 2.5 J/(cm{circumflex over ( )}3-C),thus the area specific heat for a 0.5 um thick gold layer is 1.25×10⁻⁴J/(cm{circumflex over ( )}2-C), which is ˜100 times less than that of 30um water layer, and is negligible. The negligible area specific heat ofthe Au is due to its thin thickness.

If, in a RHC card embodiment, the relevant sample is sandwiched betweentwo plates of 25 um thick each and the heating/cooling layer is 0.5 umthick, then the sample to non-sample thermal mass ratio for this case is˜1.4. Namely, when the heat losses by thermal conduction and radiationare neglected, the useful energy to the wasted energy ratio is ˜1.4, andthe useful energy to the total heating energy ratio is 58%.

In some embodiments, the sample to non-sample thermal mass ratio (NSTMratio) is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 1, 1.5, 2, 3, 4, 5, 10, 20,30, 40, 50, 60, 70, 100, 200, 300, 1000, 4000, or in a range between anyof the two values.

In preferred embodiments, the sample to non-sample thermal mass ratio(NSTM ratio) is in a range of between 0.1 to 0.2, 0.2 to 0.5, 0.5 to0.7, 0.7 to 1, 1 to 1.5, 1.5 to 5, 5 to 10, 10 to 30, 30 to 50, 50 to100, 100 to 300, 300 to 1,000, or 1,000 to 4,000.

To make the sample to non-sample thermal mass ratio high, one needs tokeep the area thermal mass of the non-sample low, which in turn, needsto make the plates and the heating/cooling layer thin, and/or the volumespecific heat low.

To make the thermal mass ratio large, one embodiment uses a thinmaterial that has multi-layers or mixed materials. For examples, acarbon fiber layer(s) with plastic sheets or carbon mixed with plastics,which can have a thickness of 0.1 um, 0.2 um, 0.5 um, 1 um, 2 um, 5 um,10 um, 25 um, 50 um, or in a range between any of the two values.

D. Thin Thickness and Large Lateral to Vertical Size Ratio (LVS Ratio)for Relevant Sample

The term of “lateral to vertical size ratio for sample” or “LVS ratiofor sample” refers to the ratio of the average lateral size of therelevant sample volume to its average vertical size. A larger LVS ratiofor sample can reduce the wasted heating energy and increase heatingspeed and/or cooling speed in the embodiments that the heating and/orcooling is primarily from the vertical direction, and can reduce thelateral thermal conduction loss at the edge of the relevant samplerelative to the total thermal energy. All of these can increase and/orcan increase cooling time.

In some embodiments, the LVS ratio for relevant sample is 5, 10, 20, 50,70, 100, 200, 300, 400, 500, 600, 700, 800, 800, 1,000, 2,000, 5000,10,000, 100,000, or in a range between any of the two values.

In some preferred embodiments, the LVS ratio for relevant sample is in arange of 5 to 10, 10 to 50, 50 to 100, 100 to 500, 500 to 1,000, 1000,to 10,000, or 10,000 to 100,000,

For example, a sample has a lateral dimension of 15 mm and a thicknessof 30 um, hence an LVS for the sample of 500.

In certain embodiments, the thickness of the relevant sample is reduced(which also can help sample heating speed), and the relevant sample hasa thickness of 0.05 um, 0.1 um, 0.2 um, 0.5 um, 1 um, 2 um, 5 um, 10 um,20 um, 30 um, 40 um, 50 um, 60 um, 70 um, 80 um, 90 um, 100 um, 200 um,300 um, or in a range between any of the two values.

In some preferred embodiments, the relevant sample has a thickness in arange between 0.05 um and 0.5 um, 0.5 um and 1 um, 1 um and 5 um, 5 umand 10 um, 10 um and 30 um, 30 um and 50 um, 50 um and 70 um, 70 um and100 um, 100 um and 200 um, or 200 um and 300 um.

E. Thin Thickness and Large Lateral to Vertical Size Ratio (LVS Ratio)for Non-Samples

The term of “lateral to vertical size ratio for non-sample” or “LVSratio for non-sample” refers to the ratio of the average lateral size ofthe portions of the two plates that sandwich the relevant sample (whichis the same as the average lateral size of the relevant sample volume)to its thickness. A large LVS ratio for non-sample can reduce thelateral thermal conduction loss at the edge of the non-sample relativeto the total thermal energy.

In some embodiments, the LVS ratio for non-sample is 5, 10, 20, 50, 70,100, 200, 300, 400, 500, 600, 700, 800, 800, 1,000, 2000, 5000, 10,000,100,000, or in a range between any of the two values.

In preferred embodiments, the LVS ratio for non-sample is in a range of5 to 10, 10 to 50, 50 to 100, 100 to 500, 500 to 1,000, 1000, to 10,000,or 10,000 to 100,000,

For example, two 25 um thick plates sandwich a sample of 5 mm or largerlateral dimension of the relevant sample, hence an LVS for thenon-sample of 200 or higher for each plate.

To shorten heating time, reduce heating energy, and increase energyefficiency, the lateral thermal conduction through a non-sample material(on the sample holder) should be reduced.

In particularly, when the first and the second plates are made of thematerials that are not good thermal materials, the thickness of theplates should be minimized.

In some embodiments, the first plate or the second plate or each of bothplates has a thickness of 10 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm,1 um, 2.5 um, 5 um, 10 um, 25 um, 50 um, 100 um, 200 um, or 500 um, 1000um, or in a range between any of the two values.

In some preferred embodiments, the first plate or the second plate oreach of both plates has a thickness of 10 nm, 100 nm, 200 nm, 300 nm,400 nm, 500 nm, 1 um, 2.5 um, 5 um, 10 um, 25 um, 50 um, 75 um, or in arange between any of the two values.

The first plate and the second plate can have the same thickness or adifferent thickness, and can be made of the same materials or differentmaterials.

In some preferred embodiments, the first plate or the second plate oreach of both plates has a thickness in a range of between 10 nm and 500nm, 500 nm and 1 um, 1 um and 2.5 um, 2.5 um and 5 um, 5 um and 10 um,10 um and 25 um, 25 um and 50 um, 50 um and 100 um, 100 um and 200 um,or 200 um and 500 um, or 500 um and 1,000 um.

In some preferred embodiments, the first plate and second plates areplastic, a thin glass, or a material with similar physical properties.The first plate or second plate has a thickness of 100 nm, 500 nm, 1 um,5 um, 10 um, 25 um, 50 um, 100 um, 175 um, 250 um, or in a range betweenany of the two values.

In some preferred embodiments, the first plate and second plates areplastic, a thin glass, or a material with similar physical properties.The first plate has a thickness of 5 um, 10 um, 25 um, 50 um, or in arange between any of the two values; while the second plate (that platethat has heating layer or cooling layer) has a thickness of 100 nm, 500nm, 1 um, 5 um, 10 um, in a range between any of the two values.

F. Cooling Layer of High K and/or High Thermal Conductivity-to-CapacityRatio (KC Ratio)

Since any thermal conduction through a non-sample material will wasteenergy and since lateral thermal conduction has much longer thermal paththan vertical thermal conduction, the energy wasted in lateral thermalconduction in non-sample materials should be minimized. One way tominimize this type of wasted energy is to use a high thermal conduction(high-K) or more precisely a high thermal conductivity-to-capacity ratio(KC ratio) materials for the cooling layer. For a given thermalconductivity, a given temperature change, and a given geometry, a high Kand/or a high KC ratio material would need much less energy to be heatedup than a low K and/or low KC ratio material.

In some embodiments, the KC ratio materials for the cooling layer isequal to or higher than 0.1 cm²/sec, 0.2 cm²/sec, 0.3 cm²/sec, 0.4cm²/sec, 0.5 cm²/sec, 0.6 cm²/sec, 0.7 cm²/sec, 0.8 cm²/sec, 0.9cm²/sec, 1 cm²/sec, 1.1 cm²/sec, 1.2 cm²/sec, 1.3 cm²/sec, 1.4 cm²/sec,1.5 cm²/sec, 1.6 cm²/sec, 2 cm²/sec, 3 cm²/sec, or in a range betweenany of the two values.

In some preferred embodiments, the KC ratio for the cooling layer is ina range of between 0.5 cm²/sec and 0.7 cm²/sec, 0.7 cm²/sec and 0.9cm²/sec, 0.9 cm²/sec and 1 cm²/sec, 1 cm²/sec and 1.1 cm²/sec, 1.1cm²/sec and 1.3 cm²/sec, 1.3 cm²/sec and 1.6 cm²/sec.

In some embodiments, a high thermal conductivity (i.e. high-K) materialis used for the cooling layer, and the high-K material has a thermalconductivity that is equal to or larger than 50 W/(m·K), 80 W/(m·K), 100W/(m·K), 150 W/(m·K), 200 W/(m·K), 250 W/(m·K), 300 W/(m·K), 350W/(m·K), 400 W/(m·K), 450 W/(m·K), 500 W/(m·K), 600 W/(m·K), 1000W/(m·K), 5000 W/(m·K), or in a range between any of the two values.

In some preferred embodiments, a high thermal conductivity (i.e. high-K)material is used for the cooling layer, and the high-K material has athermal conductivity that is in the range of 50 W/(m·K) to 100 W/(m·K),110 W/(m·K) to 200 W/(m·K), 200 W/(m·K) to 400 W/(m·K), 400 W/(m·K) to600 W/(m·K), or 400 W/(m·K) to 5000 W/(m·K).

In some embodiments, the high-K material is selected from metals,semiconductors, and allows of thermal conductivity higher than 50W/(m·K), and any combinations (including any mixtures). In someembodiments, the high-K material is selected from gold, copper, silver,and aluminum, and any combinations (including any mixtures). In someembodiments, the high-K material is selected from carbon particles,carbon tubes, graphite, silicon, and any combinations (including anymixtures).

G-1. Cooling Zone Area Larger than Lateral Relevant Sample Area andHeating Zone Area

To effectively cool a sample while reducing the wasted energy innon-sample materials, in some embodiments, a high K and/or a high KCratio material (termed “high K material”) is used as the major channelfor removing the heat from the sample. The area of high-K cooling zone(layer) should be larger than the relevant sample lateral size.

In certain embodiments, the cooling zone (layer) has an area that islarger than the lateral area of the relevant sample by a factor of 1.5,2, 3, 4, 5, 10, 20, 50, 70, 100, 200, 300, 400, 500, 600, 700, 800, 800,1,000, 2,000, 5,000, 10,000, 100,000, or in a range between any of thetwo values.

In preferred embodiments, the cooling zone (layer) has an area that islarger than the lateral area of the relevant sample by a factor in arange of 1.5 to 5, 5 to 10, 10 to 50, 50 to 100, 100 to 500, 500 to1,000, 1000, to 10,000, or 10,000 to 100,000.

To increase the cooling speed and thermal cycling efficiency, in certainembodiments, the high-K cooling layer (zone) should an area to largethan the heating zone area.

In some embodiments, the area of the cooling zone (layer) is larger thanthe area of the heating zone (layer) by a factor (i.e. the ratio of thecooling zone area to the heating zone area, “CH ratio”) of 1.1, 1.5, 2,3, 4, 5, 10, 20, 30, 40, 50, 70, 100, 200, 300, 400, 500, 600, 700, 800,800, 1,000, 5000, 10,000, 100,000, or in a range between any of the twovalues.

In preferred embodiments, the cooling zone (layer) has an area that islarger than the lateral area of the hearing zone (layer) by a factor ina range of 1.1 to 1.5, 1.5 to 5, 5 to 10, 10 to 50, 50 to 100, 100 to500, 500 to 1,000, 1,000, to 10,000, or 10,000 to 100,000.

G-2. Cooling Zone Area and Heating Zone Area are the Same as LateralRelevant Sample Area

In certain embodiments, cooling zone area and heating zone area are thesame as lateral relevant sample area, which is much smaller than thetotal sample area on the plat, and is smaller than the area of theplate. The cooling zone has an area of 1 mm², 1 mm², 1 mm², 1 mm², 1mm², 1 mm², 1 mm², 1 mm², 1 mm², 1 mm², 1 mm², 1 mm², 1 mm², 1 mm²,

The cooling zone can have different shape. In certain embodiments, thereare more than one cooling zones on one plate, and the cooling zones areseparated from each other by a low thermal conductive material such asair or plastic.

H. Heating Zone of High K and/or High Thermal Conductivity-to-CapacityRatio (KC Ratio)

Since any thermal conduction through a non-sample material that willwaste energy and lateral thermal conduction has much longer thermal paththan vertical thermal conduction, the energy wasted in lateral thermalconduction in non-sample materials should be minimized. One way tominimize this type of wasted energy is to use high thermalconductivity-to-capacity (KC) ratio materials for the materials inheating zone, which would need much less energy of heating up for agiven thermal conductivity, a given temperature change, and a givengeometry.

In some embodiments, the KC ratio materials for the heating layer isequal to or higher than 0.1 cm{circumflex over ( )}2/sec, 0.2cm{circumflex over ( )}2/sec, 0.3 cm{circumflex over ( )}2/sec, 0.4cm{circumflex over ( )}2/sec, 0.5 cm{circumflex over ( )}2/sec, 0.6cm{circumflex over ( )}2/sec, 0.7 cm{circumflex over ( )}2/sec, 0.8cm{circumflex over ( )}2/sec, 0.9 cm{circumflex over ( )}2/sec, 1cm{circumflex over ( )}2/sec, 1.1 cm{circumflex over ( )}2/sec, 1.2cm{circumflex over ( )}2/sec, 1.3 cm{circumflex over ( )}2/sec, 1.4cm{circumflex over ( )}2/sec, 1.5 cm{circumflex over ( )}2/sec, 1.6cm{circumflex over ( )}2/sec, 2 cm{circumflex over ( )}2/sec, 3cm{circumflex over ( )}2/sec, or in a range between any of the twovalues.

In some preferred embodiments, the KC ratio for the heating layer is ina range of between 0.5 cm{circumflex over ( )}2/sec and 0.7cm{circumflex over ( )}2/sec, 0.7 cm{circumflex over ( )}2/sec and 0.9cm{circumflex over ( )}2/sec, 0.9 cm{circumflex over ( )}2/sec and 1cm{circumflex over ( )}2/sec, 1 cm{circumflex over ( )}2/sec and 1.1cm{circumflex over ( )}2/sec, 1.1 cm{circumflex over ( )}2/sec and 1.3cm{circumflex over ( )}2/sec, 1.3 cm{circumflex over ( )}2/sec and 1.6cm{circumflex over ( )}2/sec, 1.6 cm{circumflex over ( )}2/sec and 2cm{circumflex over ( )}2/sec, or 2 cm{circumflex over ( )}2/sec and 3cm{circumflex over ( )}2/sec.

In some embodiments, a high thermal conductivity (i.e. high-K) materialis used for the heating layer, and the high-K material has a thermalconductivity that is equal to or larger than 50 W/(m·K), 80 W/(m·K), 100W/(m·K), 150 W/(m·K), 200 W/(m·K), 250 W/(m·K), 300 W/(m·K), 350W/(m·K), 400 W/(m·K), 450 W/(m·K), 500 W/(m·K), 600 W/(m·K), 1000W/(m·K), 5000 W/(m·K), or in a range between any of the two values.

In some preferred embodiments, a high thermal conductivity (i.e. high-K)material is used for the heating layer, and the high-K material has athermal conductivity that is in the range of 50 W/(m·K) to 100 W/(m·K),110 W/(m·K) to 200 W/(m·K), 200 W/(m·K) to 400 W/(m·K), 400 W/(m·K) to600 W/(m·K), or 400 W/(m·K) to 5000 W/(m·K).

In some embodiments, the high-K material is selected from metals,semiconductors, and allows of thermal conductivity higher than 50W/(m·K), and any combinations (including any mixtures). In someembodiments, the high-K material is selected from gold, copper, silver,and aluminum, and any combinations (including any mixtures). In someembodiments, the high-K material is selected from carbon particles,carbon tubes, graphite, silicon, and any combinations (including anymixtures).

To receive light energy by a heating zone (layer), a thermal radiationenhancement surface(s) will be used (on one side or both side of theheating zone). A thermal radiation absorption enhancement surface can beachieved by directly modify the structures of the surface (e.g.patterning nanostructures), coating a high thermal radiation materials(e.g. coating a black paint), or both.

The thermal radiation enhancement surface has a high average lightabsorptance (e.g. the black paint used in our experiments). In certainembodiments, the heating zone has a surface that has an average lightabsorptance of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, or in arange between any of the two values.

In certain preferred embodiments, the heating zone has a surface thathas an average light absorptance in a range of 30% to 40%, 40% to 60%,60% to 80% to 90%, or 90% to 100%.

In some preferred embodiments, the heating zone has a surface that hasan average light absorptance in a range of 30% to 100%, 50% to 100%, 70%to 100%, or 80% to 100%.

In certain embodiments, the heating zone has a surface that has anaverage light absorptance of a value given above by averaging over awavelength range 400 nm to 800 nm, 700 nm to 1500 nm, 900 nm to 2000 nm,or 2000 nm to 20000 nm.

Increasing Thermal Radiative Cooling

In certain embodiments, a fast temperature cycling is achieved byincreasing thermal radiative cooling percentage in the total cooling ofthe sample and the sample holder (i.e. removing heat to the environment)during a thermal cycling, preferably through using high thermalconductivity material as the material for thermal radiative cooling. Onereason is that cooling through lateral thermal conduction needs to heatup many non-sample materials, wasting energy. Another reason is thatthermal radiation cooling is proportional to the fourth power of thetemperature and can be more effective than thermal conduction in a thinfilm.

To enhancing thermal radiative cooling, in certain embodiments, thethermal radiative cooling uses a cooling layer (cooling zone) that isenhanced for thermal radiative cooling. The enhancement includes (i)increase thermal conductivity of the cooling zone (layer), (ii)enlarging the area of the cooling zone (layer), (iii) enhance thesurface thermal radiation of the cooling zone, and (iv) a combinationthereof.

Examples of a high thermal conductivity materials are metals (such asgold, silver, coper, aluminum), semimetals, semiconductors (e.g.silicon) or a combination thereof.

To further enhance thermal radiation of a cooling zone (layer), athermal radiation enhancement surface(s) will be used (on one side orboth side of the cooling zone). A thermal radiation enhancement surfacecan be achieved by directly modify the structures of the surface (e.g.patterning nanostructures), coating a high thermal radiation material(e.g. coating a black paint), or both.

The thermal radiation enhancement surface has a high average lightabsorptance (e.g. the black paint used in our experiments). In certainembodiments, the cooling zone has a surface that has an average lightabsorptance of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, or in arange between any of the two values.

In certain preferred embodiments, the cooling zone has a surface thathas an average light absorptance in a range of 30% to 40%, 40% to 60%,60% to 80% to 90%, or 90% to 100%.

In some preferred embodiments, the cooling zone has a surface that hasan average light absorptance in a range of 30% to 100%, 50% to 100%, 70%to 100%, or 80% to 100%.

In certain embodiments, the cooling zone has a surface that has anaverage light absorptance of a value given above by averaging over awavelength range 400 nm to 800 nm, 700 nm to 1,500 nm, 900 nm to 2,000nm, or 2,000 nm to 20,000 nm.

In certain embodiments, the surface thermal radiation enhancement layeris black paint, plasmonic structures, nanostructures, or any combinationthereof.

The high thermal radiation materials are polymer mixtures that lookblack by human eyes (often termed “black paints”). A high thermalradiation material include, but not limited to, a mixture of polymersand nanoparticles. One example of the nanoparticles is black carbonnanoparticle, carbon, nanotubes, graphite particles, graphene, metalnanoparticles, semiconductor nanoparticles, or a combination thereof.

The high thermal radiation material further comprises a material that isdeposited or made on the layer surface and look blacks by human eyes.The materials include, but not limited to, black carbon nanoparticle,carbon, nanotubes, graphite particles, graphene, metal nanoparticles,semiconductor nanoparticles, or a combination thereof.

The plasmonic structures include nanostructured plasmonic structures.

In some embodiments, a cooling layer comprise a layer of high thermalconductivity metal (50 W/(m·K) or higher) with a surface thermalradiation enhancement layer. In some embodiments, the surface thermalradiation enhancement layer has a low lateral thermal conductance, whichis due to either ultrathin layer, low thermal conductivity, or both.

Percentage of Thermal Radiative Cooling.

In certain embodiments, thermal radiative cooling is achieved byincreasing the area of radiative cooling layer (i.e. a high-K material,unless stated otherwise), and the radiative cooling layer area is largerthan the lateral area of the relevant sample by a factor of 1.2, 1.5, 2,3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80 100, 200, 300, 400, 500, 600,700, 800, 800, 1,000, 2,000, 5,000, 10,000, 100,000, or in a rangebetween any of the two values.

In preferred embodiments, the radiative cooling zone (layer) has an areathat is larger than the lateral area of the relevant sample by a factorin a range of 1.2 to 3, 3 to 5, 5 to 10, 10 to 50, 50 to 100, 100 to500, 500 to 1,000, 1,000, to 10,000, or 10,000 to 100,000.

In some embodiments, the ratio of the thermal radiation cooling by thecooling zone (layer) to the total cooling of the sample and sampleholder during a thermal cycling is 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 99%, or in a range between any of the two values.

In some preferred embodiments, the ratio of the thermal radiationcooling by the cooling zone (layer) to the total cooling of the sampleand sample holder during a thermal cycling is in a range of between 10%and 20%, 20% and 30%, 30% and 40%, 40% and 50%, 50% and 60%, 60% and70%, 70% and 80%, 80% and 90%, or 90% and 99%.

J. Control of Cooling Layer Thickness

In certain embodiments, the thickness of the cooling layer thickness isconfigured to facilitate to optimize heating locally and/or energyefficiency. If the cooling zone (layer) is too thick, a significantpercentage of the heating energy will be wasted by the cooling layer,lengthening heating time (for a given heating power). On the other hand,if the cooling zone is too thin, the cooling time will be significantlylonger. Hence, the cooling layer thickness should be optimized for bothfast heating and cooling.

Through our experiments, we found that the thickness of the high-Kcooling layer can regulate the cooling rate. By selecting a properhigh-K cooling layer thickness and a proper LED power density, a fastheating and cooling can be achieved.

Since a thermal conductance of a layer proportional to a material'sthermal conductivity times the layer thickness, so it is this productshould be optimized.

In some embodiments, a cooling zone (layer) has thermal conductivitytimes its thickness of 6×10⁻⁵ W/K, 9×10⁻⁵ W/K, 1.2×10⁻⁴ W/K, 1.5×10⁻⁴W/K, 1.8×10⁻⁴ W/K, 2.1×10⁻⁴ W/K, 2.7×10⁻⁴ W/K, 3×10⁻⁴ W/K, 1.5×10⁻⁴ W/K,or in a range between any of the two values.

In some preferred embodiments, a cooling zone (layer) has thermalconductivity times its thickness in a range of 6×10⁻⁵ W/K to 9×10⁻⁵ W/K,9×10⁻⁵ W/K to 1.5×10⁻⁴ W/K, 1.5×10⁻⁴ W/K to 2.1×10⁻⁴ W/K, 2.1×10⁻⁴ W/Kto 2.7×10⁻⁴ W/K, 2.7×10⁻⁴ W/K to 3×10⁻⁴ W/K, or 3×10⁻⁴ W/K to 1.5×10⁻⁴W/K.

In certain preferred embodiments, a cooling zone (layer) has thermalconductivity times its thickness in a range of 9×10⁻⁵ W/K to 2.7×10⁻⁴W/K, 9×10⁻⁵ W/K to 2.4×10⁻⁴ W/K, 9×10⁻⁵ W/K to 2.1×10⁻⁴ W/K, or 9×10⁻⁵W/K to 1.8×10⁻⁴ W/K.

In one embodiment, a cooling zone comprises a gold layer of a thicknessin the range of 200 nm to 800 nm. In another embodiment, a cooling zonecomprises a gold layer of a thickness in the range of 300 nm to 700 nm.

K. Large Conductance Between Sample and Heating Zone or Cooling Zone

For a fast heating and cooling a sample, the thermal conduction per unitarea between a relevant sample and a heating layer and/or the coolinglayer should be large. The thermal conduction per area is equal to theconductivity (unit volume) divided by the material thickness for thematerials that are between the HC layer and the sample. For example, for100 nm thick of PS as the second plate which has the HC layer on onesurface and the sample on the other surface, the conductance between theHC layer and the sample is ˜1000 W/(m²·K)

Based on experiments, in some embodiments of a RHC card, the materialsbetween the heating zone and the relevant sample has a thermalconductivity and a thickness configured to be about 1000 W/(m²·K) orhigher.

In some embodiments of a RHC card, the materials between the heatingzone and the relevant sample has a thermal conductivity and a thicknessconfigured to have a conductance per unit area that is equal to orlarger than 1000 W/(m²·K), 2000 W/(m²·Km²·K), 3000 W/(m²·Km²·K), 4000W/(m²·Km²·K), 5000 W/(m²·Km²·K), 7000 W/(m²·Km²·K), 10000 W/(m²·K),20000 W/(m²·K), 50000 W/(m²·K), 50000 W/(m²·K), 100000 W/(m²·K), or in arange of any the values.

A preferred conductance per unit area of the material between theheating zone and the relevant sample is in a range of 1000 W/(m²·K) to2000 W/(m²·K), 2000 W/(m²·K) to 4000 W/(m²·K), 4000 W/(m²·K) to 10,000W/(m²·K), or 10000 W/(m²·K) to 100000 W/(m²·K).

In another preferred embodiment, it has zero distance between theheating zone and the relevant sample, and hence an infinity for theconductance per unit area of the material between the heating zone andthe relevant sample.

In certain embodiments, the heating layer or the cooling layer isseparated from a relevant sample by a thin plastics plate (or film)which has a thermal conductivity in the range of 0.1 to 0.3 W/(m·K), andthe thin plastic layer has a thickness of 0 nm, 10 nm, 50 nm, 100 nm,200 nm, 300 nm, 400 nm, 500 nm, 1 um, 2.5 um, 5 um, 10 um, 25 um, 50 um,75 nm 100 um, 150 um, or in a range between any of the two values

In some preferred embodiments, the thin plastic plate (or film) thatseparate the relevant sample from the heating layer or the cooling layerhas thickness in a range between 0 nm and 100 nm, 100 nm and 500 nm, 500nm and 1 um, 1 um and 5 um, 5 um and 10 um, um and 25 um, 25 um and 50um, 50 um and 75 um, 75 um and 100 um, or 100 um and 150 um.

In one preferred embodiment of the RHC card, the thin plastic plate (orfilm) that separate the relevant sample from the heating layer or thecooling layer has thickness of 1 nm, 10 nm, 0.1 um, 0.5 um, 1 um, 5 um,10 um, 20 um, 25 um, or a range between any two values.

L. Small Relative Reagent Lateral Diffusion

In order to make a biochemical reaction substantially uniform in therelevant sample volume during a temperature change or a thermal cycling,the average lateral area of the relevant sample should be significantlylarger than the lateral diffusion of the nucleic acids and/or otherregents used for a molecular amplification and/or reaction. In this way,during the time of temperature change or a thermal cycling, most of themolecules inside the relevant sample volume do not have enough time todiffuse out of the relevant sample volume, while most of the moleculesoutside the relevant sample volume do not have enough time to diffuseinto the relevant sample volume.

Considering a thermal cycling time duration of 3 min and a diffusionconstant of ˜1×10{circumflex over ( )}-6 cm2/s for a molecule about 600Da molecular weight, the diffusion length is ˜130 um.

In certain embodiments, the ratio of the average lateral size of therelevant sample volume to the diffusion length of the reagent during thetime for thermal cycling or a reaction is equal to or larger than 5, 6,7, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, 1000,5000, 10000, 100000, or in a range between any two values.

In some preferred embodiments, the ratio of the average lateral size ofthe relevant sample volume to the diffusion length of the reagent duringthe time for thermal cycling or a reaction is in a range of 5 to 10, 10to 30, 30 to 60, 6 to 100, 100 to 200, 200 to 500, 500 to 1000, 1000 to5000, 5000 to 10,000, or 10,000 to 100,000.

In some preferred embodiments, the ratio of the average lateral size ofthe relevant sample volume to the diffusion length of the reagent duringthe time for thermal cycling or a reaction is in a range of 5 to 10, 10to 30, 30 to 60, 6 to 100, 100 to 200, 200 to 500, 500 to 1,000, 1,000to 5,000, 5,000 to 10,000, or 10,000 to 100,000.

In certain preferred embodiments, the average lateral dimension of therelevant volume is 1 mm, 2 mm, 3 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm 10 mm,12 mm, 15 mm, 20 mm, 30 mm, 40 mm, 50 mm, 70 mm, 100 mm, 200 mm, or in arange between any two values.

In some preferred embodiments, the average lateral dimension of therelevant volume is in a range of 1 mm to 5 mm, 5 mm to 10 mm, 10 mm to20 mm, 20 mm to 40 mm, 40 mm to 70 mm, 70 mm to 100 mm, or 100 mm to 200mm.

In another preferred embodiments, the average lateral dimension of therelevant volume is in a range of 1 mm to 5 mm, 1 mm to 10 mm, or 5 mm to20 mm.

M. Without Edge Sealing or Simple Edge Sealing

To simplify the sample holder operation and cost, in certainembodiments, there is no sealing between the two plates that confine asample; namely, the sample sandwiched between the plates can evaporatefrom the sample edge into environment. However, in our experiments, wefound that in our sample card configuration, such evaporation isnegligible relative to total sample volume, due to a large ratio of thelateral sample area to the sample edge area; the plates have preventedmost of the evaporation.

In some embodiments, an enclosure ring spacer or some discontinuousspacer walls can be put on one or both of the plates to reduce oreliminate a sample evaporation.

P-2 Forced Air Cool

In certain embodiments, there is a forced air cooling/circulating systemnear the RHC card to speed up the cooling process. The example of forcedair cooling system includes but not limit to a fan circulating the coolair near the card, several fans circulating the cool air near the card,a cooling source cool the air near the card, a cooling pad direct touchthe card or their combinations.

In certain embodiments, there is a forced air cooling/circulating systemcooling the air on the top surface of the card.

In certain embodiments, there is a forced air cooling/circulating systemcooling the air on the bottom surface of the card.

In certain embodiments, there is a forced air cooling/circulating systemcooling the air surrounding all the surface of the card.

2. Mechanical Structure Designs

N. Movable Plates and Compressed Open Flow, Hinges, Opening Notches,Recessed Edge and Sliders

To load a sample simply, in certain embodiments in the presentinvention, the two plates of a RHC card are movable relative to eachother into different configurations. A sample is deposited at an openconfiguration of the plates, and then the plates are pressed into aclosed configuration. During the pressing, the sample will flow betweenthe plates into a thin layer, and the flow is termed “compressed openflow”, since there are plenty room between the plates that allow thesample to flow.

In certain embodiments, spaces for regulating the sample thickness areadded on one or both of the plates, hence a device for rapidly changingthe temperature of a fluidic sample, comprising:

a first plate (10), a second plate (20), a heating layer (112-1), and acooling layer (112-2), wherein:

the first and second plates are movable relative to each other intodifferent configurations;

each of the first plate and the second plate has, on its respectiveinner surface, a sample contact area for contacting a fluidic sample;wherein the sample contact areas face each other, are separated by anaverage separation distance of 200 um or less, and are capable ofsandwiching the sample between them;

the heating layer is:

positioned on the inner surface, the outer surface, or inside of one ofthe plates, and

configured to heat a relevant volume of the sample, wherein the relevantvolume of the sample is a portion or an entirety of the sample that isbeing heated to a desired temperature; and

the cooling layer is:

positioned on the inner surface, the outer surface, or inside of one ofthe plates;

configured to cool the relevant sample volume; and

comprises a layer of material that that has a thermal conductivity tothermal capacity ratio of 0.6 cm²/sec or larger;

wherein one of the configurations is an open configuration, in which:the two plates are partially or completely separated apart and theaverage spacing between the plates is at least 300 um;

wherein another of the configurations is a closed configuration which isconfigured after the fluidic sample is deposited on one or both of thesample contact areas in the open configuration; and in the closedconfiguration: at least part of the sample is confined by the two platesinto a layer, wherein the average sample thickness is 200 um or less;and

wherein, in some embodiments, the heating layer and cooling layer arethe same material layer that has a heating zone and a cooling zone, andwherein the heating zone and cooling zone can have the same area ordifferent areas.

In some embodiments, the sample holder (also termed “RHC card” or“Q-card”) with movable plates further comprises hinges, notches,recesses, which help to facilitate the manipulation of the sample holderand the measurement of the samples. Furthermore, the sample holders canslide into sliders. The structure, material, function, variation anddimension of the hinges, notches, recesses, sliders and compress openflow are herein disclosed, or listed, described, and summarized in PCTApplication (designating U.S.) Nos. PCT/US2016/046437 andPCT/US2016/051775, which were respectively filed on Aug. 10, 2016 andSep. 14, 2016, U.S. Provisional Application No. 62/456,065, which wasfiled on Feb. 7, 2017, U.S. Provisional Application No. 62/456,287,which was filed on Feb. 8, 2017, and U.S. Provisional Application No.62/456,504, which was filed on Feb. 8, 2017, all of which applicationsare incorporated herein in their entireties for all purposes.

Spacers (13)

In certain embodiments, the spacers as described in embodiment SH-5 willbe used to regulate the sample thickness and make the thickness uniform.The spacers also allow to achieve uniform sample thickness, even whenboth plates are very thin (e.g. 25 um thick or less).

In certain embodiments, the spacers are fixed on one or both of theplates. In certain embodiments, the spacers are mixed with the sample.In some embodiments, the spacers have a uniform height and the spacers,together with the first plate and the second plate, regulate the samplelayer. In some embodiments, the thickness of the sample layer issubstantially equal to the height of the spacers.

In some embodiments, the plates are flat (e.g. as shown in FIG. 3 ). Insome embodiments, either one or both of the plates include wells (e.g.as shown in FIG. 4 ). For example, in certain embodiments the width ofthe wells can be less than 500 um, 200 um, 100 um, 50 um, 25 um, 10 um,5 um, 2.5 um, 1 um, 500 nm, 400 nm, 300 nm, 200 nm, or 100 nm, or in arange between any of the two values. In certain embodiments, the depthof the wells can be less than 500 um, 200 um, 100 um, 50 um, 25 um, 10um, 5 um, 2.5 um, 1 um, 500 nm, 400 nm, 300 nm, 200 nm, 100 nm, 50 nm,20 nm, 10 nm, 5 nm, 2 nm, or 1 nm, or in a range between any of the twovalues

In some embodiments, one or both of the plates have wells and most orentire of the samples are only inside the well of one plate and iscovered by other plate (not shown in the figures).

P. Sample Cartridge and Thermal Conduction Isolation

In certain embodiments, the RHC card (sample holder) can be furthermounted on a sample cartridge. The cartridge can be configured to slidein or out a base (also termed “adaptor”). A base houses the powersource, temperature sensors and controllers, signal measurement devices,and a slot for the sample holder with or without a cartridge to slide inor out of the base.

In some embodiments, the sample holder, the cartridge (i.e. the sampleholder support) or both are “thermal conduction isolated”, namely, theydo not have or almost do not have, during a thermal cycling, a thermalconduction to the environment. In this case, the cooling in the thermalcycling is essentially by thermal radiation (this is termed “noconductive heat transfer”). In some embodiment, the “thermal conductionisolation” is achieved in the sample holder, the cartridge, or both byconfiguration their materials, the geometry (including of thicknessreduction), or both.

Q. Combination of Above

An embodiment of a RHC card can be any combination of the specificationdescribed in SH-1, SH-2, SH-3 and in subsections of A to P.

R. Heating Sources

The heating layer or the heating/cooling layer in a RHC card isconfigured to be heated by a heating source, wherein the heating sourcedelivers heat energy to the heating/cooling layer optically,electrically, by radio frequency (RF) radiation, or a combinationthereof.

S. Base (i.e. Adaptor)

In some embodiments, the apparatus further comprises a base (an adaptor)that is configured to house the sample card, the heating source,temperature sensors, a part of an entire of temperature controlled(include a smartphone in some embodiments), extra-heat sink(optionally), a fan (optionally) or a combination of thereof. In someembodiments, the adaptor comprises a card slot, into which the samplecard or a sample cartridge can be inserted. In some embodiments, thesample card or the sample cartridge, after being fully inserted into theslot, or after reaching a pre-defined position in the slot, isstabilized and stays in place without any movement.

T. Smartphone

In some embodiments, a smartphone is used to mage the sample card,controlling the heating and/cooling, sensing a signal, monitor operationuse camera, provide light/energy with a flash, communicate to a local ora remote device, integrated through a base (adaptor) in a system, of acombination thereof.

U. Applications for Isothermal Nucleic Acid Amplification

The present invention with a slight modification also provides usefuldevices and methods for isothermal nucleic acid amplification, where asample temperature needs to be raised from environment to an elevatedtemperature (i.e. 65° C.) and keep at the temperature for a period oftime (i.e. 5-10 min). In some embodiments, one of the modificationsneeded for isothermal nucleic acid amplification test, is to reduce oreliminate the cooling zone/layer, so that loss of thermal energy fromthe sample and/or the sample holder to the environment is reduced.

The present invention with a slight modification provides useful devicesand methods for reverse transcription polymerase chain reaction, whichcontains an isothermal process before the regular PCR, where a sampletemperature needs to be raised from environment to an elevatedtemperature (i.e. 50° C.) and keep at the temperature for a period oftime (i.e. 5-10 min). The present invention with a slight modificationprovides useful devices and methods for minimize PCR cross-contaminationas method to use dUTP and uracil-DNA N-glycosylase, where a sampletemperature needs to be raised from environment to an elevatedtemperature (i.e. 50° C.) and keep at the temperature for a period oftime (i.e., 1-20 min).

Sample Wells

In certain embodiments, one or both of the plates have sample wells,wherein the well regulates the maximum volume of the sample in the welland prevents the sample to flow into other location of the plates.

Plate Thickness

To reduce the thermal mass of the first and second plates as well asreduce the lateral thermal conduction loss in the plates, the thicknessof the first plate and the second plate is preferred to be thin.

In certain embodiments, the first plate or the second plate has athickness of 2 nm or less, 10 nm or less, 100 nm or less, 200 nm orless, 500 nm or less, 1000 nm or less, 2 μm (micron) or less, 5 μm orless, 10 μm or less, 20 μm or less, 50 μm or less, 100 μm or less, 150μm or less, 200 μm or less, 300 μm or less, 500 μm or less, 800 μm orless, 1 mm (millimeter) or less, 2 mm or less, 3 mm or less, 5 mm orless, 10 mm or less, or in a range between any two of these values.

In some embodiments, the first plate or the second plate has a thicknessof 10 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 1 um, 2.5 μm, 5 um, 10um, 25 um, 50 um, 100 um, 200 um, or 500 um, 1000 um, or in a rangebetween any of the two values.

The first plate and the second plate can have the same thickness or adifferent thickness, and can be made of the same materials or differentmaterials.

In some preferred embodiments, the first plate or the second plate has athickness in a range of between 10 nm and 500 nm, 500 nm and 1 um, 1 umand 2.5 μm, 2.5 um and 5 um, 5 um and 10 um, 10 um and 25 um, 25 um and50 um, 50 um and 100 um, 100 um and 200 um, or 200 um and 500 um, or 500um and 1000 um.

A preferred thickness of the first plate or the second plate is 10 nm orless, 100 nm or less, 200 nm or less, 500 nm or less, 1000 nm or less, 2μm (micron) or less, 5 μm or less, 10 μm or less, 20 μm or less, 50 μmor less, 100 μm or less, 150 μm or less, 200 μm or less, 300 μm or less,500 μm or less, or in a range between any two of the values.

In some preferred embodiments, the thickness of the plate that has theheating/cooling layer is thinner than the other plate that does not havea heater.

In some preferred embodiments, the first plate has a thickness of 100nm, 200 nm, 500 nm, 1 μm (micron), 2 μm, 5 μm, 10 μm, 25 μm, 50 μm, 100μm, 125 μm, 150 μm, 175 μm, 200 μm, 250 μm, or in a range between anytwo of the values; while the second plate has a thickness of 25 μm, 50μm, 100 μm, 125 μm, 150 μm, 175 μm, 200 μm, 250 μm, 500 μm, 1 mm, 1.5mm, 2 mm, or in a range between any two of the values,

In some embodiments, the average thickness for at least one of theplates is in the range of 1 to 1000 μm, 10 to 900 μm, 20 to 800 μm, 25to 700 μm, 25 to 800 μm, 25 to 600 μm, 25 to 500 μm, 25 to 400 μm, 25 to300 μm, 25 to 200 μm, 30 to 200 μm, 35 to 200 μm, 40 to 200 μm, 45 to200 μm, or 50 to 200 μm.

In some embodiments, the average thickness for at least one of theplates is in the range of 50 to 75 μm, 75 to 100 μm, 100 to 125 μm, 125to 150 μm, 150 to 175 μm, or 175 to 200 μm.

In some embodiments, the average thickness for at least one of theplates is about 50 μm, about 75 μm, about 100 μm, about 125 μm, about150 μm, about 175 μm, or about 200 μm.

Plate Area. In some embodiments, the first plate and/or the second platehas a lateral area of 1 mm² (square millimeter) or less, 10 mm² or less,25 mm² or less, 50 mm² or less, 75 mm² or less, 1 cm² (squarecentimeter) or less, 2 cm² or less, 3 cm² or less, 4 cm² or less, 5 cm²or less, 10 cm² or less, 20 cm² or less, 30 cm² or less, 50 cm² or less,100 cm² or less, 500 cm² or less, 1,000 cm² or less, 5,000 cm² or less,10,000 cm² or less, or in a range between any two of these values.

In preferred embodiments, the first plate and/or the second plate has alateral area in a range of 1 mm² (square millimeter) to 10 mm², 10 mm²to 50 mm², 50 mm² to 100 mm², 1 cm² to 5 cm², 5 cm² to 20 cm², 20 cm² to50 cm², 50 cm² to 100 cm², 100 cm² to 500 cm², 500 cm² to 1000 cm², or1000 cm² to 10,000 cm².

In some embodiments, the first plate and the second plate have the samelateral dimension. In some embodiments, one of the plates has an areathat is different from the other plates by 10% or less, 30% or less, 50%or less, 80% or less, 90% or less, 95% or less, 99% or less, or in arange between any two of these values (take the largest plate is thebase in calculation the different percentage).

In some embodiment, the first plate and/or the second plate has a widthor a length of 5 mm, 10 mm, 20 mm, 25 mm, 30 mm, 40 mm, 50 mm, 75 mm,100 mm, or in a range between any two of these values.

In preferred embodiments, the first plate and/or the second plate has awidth or a length in a range of 5 mm to 10 mm, 20 mm to 30 mm, 30 mm to50 mm, 50 mm to 75 mm, or 75 mm to 100 mm.

In one preferred embodiment, the plate has a width or length in a rangeof 5 mm to, 50 mm. In another preferred embodiment, the plate has awidth in a range of 5 mm to 50 mm and a length in a range of 6 mm to 70mm.

Materials for Plates

In some embodiments, the materials for the first plate and the secondplates, contain but are not limit to polymers (e.g. plastics) oramorphous organic materials. The polymer materials include, not limitedto, acrylate polymers, vinyl polymers, olefin polymers, cellulosicpolymers, noncellulosic polymers, polyester polymers, Nylon, cyclicolefin copolymer (COC), poly(methyl methacrylate) (PMMA), polycarbonate(PC), cyclic olefin polymer (COP), liquid crystalline polymer (LCP),polyamide (PA), polyethylene (PE), polyimide (PI), polypropylene (PP),poly(phenylene ether) (PPE), polystyrene (PS), polyoxymethylene (POM),polyether ether ketone (PEEK), polyether sulfone (PES), poly(ethylenephthalate) (PET), polytetrafluoroethylene (PTFE), polyvinyl chloride(PVC), polyvinylidene fluoride (PVDF), polybutylene terephthalate (PBT),fluorinated ethylene propylene (FEP), perfluoroalkoxyalkane (PFA),polydimethylsiloxane (PDMS), rubbers, or any combinations of thereof.

In some embodiments, the materials for the first plate and the secondplate contain but are not limit to inorganic materials includingdielectric materials of silicon oxide, porcelain, orcelain (ceramic),mica, glass, oxides of various metals, etc.

In some embodiments, the materials for the first plate and the secondplate contain but are not limit to inorganic materials includingaluminum oxide, aluminum chloride, cadmium sulfide, gallium nitride,gold chloride, indium arsenide, lithium borohydride, silver bromide,sodium chloride, graphite, carbon nanotubes, carbon fibers, etc.

In some embodiments, the materials for the first plate and the secondplate contain but are not limit to metals (e.g. gold, copper, aluminum,etc.) and alloys.

In some embodiments, the materials for the first plate and the secondplate are made of multi-layers and/or mixture of the materials listedabove.

Heating Layer and Cooling Layer

In certain embodiments, a heating layer (112-1) and a cooling layer(112-2) comprises high K material and/or a high KC ratio material. Thehigh K and/or high KC ratio material comprises materials/structures,such as, but not limited to, metallic film, semiconductors, semimetals,plasmonic surface, metamaterials (e.g. nanostructures), black silicon,graphite, carbon nanotube, silicon sandwich, graphene, superlattice,plasmonic materials, any material/structure that is capable ofefficiently absorbing the electromagnetic wave and converting theabsorbed energy into thermal energy, and any combination thereof.

For a heating layer that is heated by an optical heating source, aheating layer comprises a material layer that significantly absorb theradiated energy from the optical heating source. The significantabsorption means that the heating/cooling layer absorbs the radiatedenergy from the optical heating source more significantly than thesample and the plates.

In certain embodiments, the heating/cooling layer has thickness in therange of 50 nm to 15 um. In certain embodiments, the heating/coolinglayer comprise a high K layer that has thickness in the range of 100 nmto 1 um.

In some embodiments, the dimension of the light heating area is about 1um, 2 um, 5 um, 10 um, 20 um, 50 um, 100 um, 200 um, 500 um, 1 mm, 2 mm,5 mm, 10 mm, 20 mm, 50 mm, or 100 mm, or in a range between any of thetwo values. In various embodiments, the size and shape of the lightheating areas can vary.

In some embodiments, the heating/cooling layer comprise adot-coupled-dots-on-pillar antenna (D2PA) array, such as, but notlimited to the D2PA array described in U.S. Provisional PatentApplication No. 61/347,178, which was filed on May 21, 2010, U.S.Provisional Patent Application 61/622,226, which was filed on Apr. 10,2012, U.S. PCT Application No. PCT/US2011/037455, which was filed on May20, 2011, PCT Application No. PCT/US2013/032347, which was filed on Mar.15, 2013, and U.S. patent application Ser. No. 13/699,270, which wasfiled on Jun. 13, 2013, the complete disclosures of which are herebyincorporated by reference for all purposes.

In some embodiments, there can be more than one heating/cooling layer.For examples, at least two surfaces of any of the first or second plateshave a heating/cooling layer.

In some embodiments, the heating/cooling layer can be two-layermaterials: one layer for heating and one for cooling, and the two-layermaterials can be on the same surface of any of the first or secondplate. For sample, the heating layer can be on the outer surface of thesecond plate, while the cooling layer is on the outer surface or theinner surface of the first plate. Even the cooling layer is on the outersurface of the first plate, which should be efficient in cooling thesample as long as the first plate has thin thickness (e.g., 25 um orless).

Spacers

In some embodiments of the present invention there are spacers betweenthe two plates. In some embodiments, at least one of the spacers is inthe sample contact area. In some embodiments, the spacers have uniformheight. In some embodiments, the thickness of the sample is the sampleas the height of the spacers. In some embodiments, the spacers are fixedon one of the plates.

Spacers' Function. In present invention, the spacers are configured tohave one or any combinations of the following functions and properties:the spacers are configured to (1) control, together with the plates, thethickness of the sample or a relevant volume of the sample (Preferably,the thickness control is precise, or uniform or both, over a relevantarea); (2) allow the sample to have a compressed regulated open flow(CROF) on plate surface; (3) not take significant surface area (volume)in a given sample area (volume); (4) reduce or increase the effect ofsedimentation of particles or analytes in the sample; (5) change and/orcontrol the wetting propertied of the inner surface of the plates; (6)identify a location of the plate, a scale of size, and/or theinformation related to a plate, or (7) do any combination of the above.

Spacer architectures and shapes. To achieve desired sample thicknessreduction and control, in certain embodiments, the spacers are fixed onits respective plate. In general, the spacer can have any shape, as longas the spacers are capable of regulating the sample thickness during aCROF process, but certain shapes are preferred to achieve certainfunctions, such as better uniformity, less overshoot in pressing, etc.

The spacer(s) is a single spacer or a plurality of spacers. (e.g. anarray). Some embodiments of a plurality of spacers is an array ofspacers (e.g. pillars), where the inter-spacer distance is periodic oraperiodic, or is periodic or aperiodic in certain areas of the plates,or has different distances in different areas of the plates.

There are two kinds of the spacers: open-spacers and enclosed-spacers.The open-spacer is the spacer that allows a sample to flow through thespacer (i.e. the sample flows around and pass the spacer. For example, apost as the spacer), and the enclosed spacer is the spacer that stop thesample flow (i.e. the sample cannot flow beyond the spacer. For example,a ring shape spacer and the sample is inside the ring). Both types ofspacers use their height to regular the final sample thickness at aclosed configuration.

In some embodiments, the spacers are open-spacers only. In someembodiments, the spacers are enclosed-spacers only. In some embodiments,the spacers are a combination of open-spacers and enclosed-spacers.

The term “pillar spacer” means that the spacer has a pillar shape andthe pillar shape refers to an object that has height and a lateral shapethat allow a sample to flow around it during a compressed open flow. Insome embodiments, the spacers have a flat top (e.g. pillars with a flattop to contact a plate).

In some embodiments, the lateral shapes of the pillar spacers are theshape selected from the groups of (i) round, elliptical, rectangles,triangles, polygons, ring-shaped, star-shaped, letter-shaped (e.g.L-shaped, C-shaped, the letters from A to Z), number shaped (e.g. theshapes like 0 1, 2, 3, 4, . . . to 9); (ii) the shapes in group (i) withat least one rounded corners; (iii) the shape from group (i) withzig-zag or rough edges; and (iv) any superposition of (i), (ii) and(iii). For multiple spacers, different spacers can have differentlateral shape and size and different distance from the neighboringspacers.

In some embodiments, the spacers can be and/or can include posts,columns, beads, spheres, and/or other suitable geometries. The lateralshape and dimension (i.e., transverse to the respective plate surface)of the spacers can be anything, except, in some embodiments, thefollowing restrictions: (i) the spacer geometry will not cause asignificant error in measuring the sample thickness and volume; or (ii)the spacer geometry would not prevent the out-flowing of the samplebetween the plates (i.e. it is not in enclosed form). But in someembodiments, they require some spacers to be closed spacers to restrictthe sample flow.

In some embodiments, the shapes of the spacers have rounded corners. Forexample, a rectangle shaped spacer has one, several or all cornersrounded (like a circle rather 90 degree angle). A round corner oftenmake a fabrication of the spacer easier, and in some cases less damageto a biological material.

The sidewall of the pillars can be straight, curved, sloped, ordifferent shaped in different section of the sidewall. In someembodiments, the spacers are pillars of various lateral shapes,sidewalls, and pillar-height to pillar lateral area ratio.

In a preferred embodiment, the spacers have shapes of pillars forallowing open flow.

Spacers' materials. In the present invention, the spacers are generallymade of any material that is capable of being used to regulate, togetherwith the two plates, the thickness of a relevant volume of the sample.In some embodiments, the materials for the spacers are different fromthat for the plates. In some embodiments, the materials for the spacesare at least the same as a part of the materials for at least one plate.

The spacers are made a single material, composite materials, multiplematerials, multilayer of materials, alloys, or a combination thereof.Each of the materials for the spacers is an inorganic material, amorganic material, or a mix, wherein examples of the materials are givenin paragraphs of Mat-1 and Mat-2. In a preferred embodiment, the spacersare made in the same material as a plate used in CROF.

Spacer's mechanical strength and flexibility. In some embodiments, themechanical strength of the spacers are strong enough, so that during thecompression and at the closed configuration of the plates, the height ofthe spacers is the same or significantly same as that when the platesare in an open configuration. In some embodiments, the differences ofthe spacers between the open configuration and the closed configurationcan be characterized and predetermined.

The material for the spacers is rigid, flexible or any flexibilitybetween the two. The rigid is relative to a give pressing forces used inbringing the plates into the closed configuration: if the space does notdeform greater than 1% in its height under the pressing force, thespacer material is regarded as rigid, otherwise a flexible. When aspacer is made of material flexible, the final sample thickness at aclosed configuration still can be predetermined from the pressing forceand the mechanical property of the spacer.

Spacer inside Sample. To achieve desired sample thickness reduction andcontrol, particularly to achieve a good sample thickness uniformity, incertain embodiments, the spacers are placed inside the sample, or therelevant volume of the sample. In some embodiments, there are one ormore spacers inside the sample or the relevant volume of the sample,with a proper inter spacer distance. In certain embodiments, at leastone of the spacers is inside the sample, at least two of the spacersinside the sample or the relevant volume of the sample, or at least of“n” spacers inside the sample or the relevant volume of the sample,where “n” can be determined by a sample thickness uniformity or arequired sample flow property during a CROF.

Spacer height. In some embodiments, all spacers have the samepre-determined height. In some embodiments, spacers have differentpre-determined height. In some embodiments, spacers can be divided intogroups or regions, wherein each group or region has its own spacerheight. And in certain embodiments, the predetermined height of thespacers is an average height of the spacers. In some embodiments, thespacers have approximately the same height. In some embodiments, apercentage of number of the spacers have the same height. In someembodiments, on the same plate, the spacer height in one ration isdifferent from the spacer height in another region. In some cases, theplate with different spacer height in different regions have advantagesof assaying.

The height of the spacers is selected by a desired regulated finalsample thickness and the residue sample thickness. The spacer height(the predetermined spacer height) and/or sample thickness is 3 nm orless, 10 nm or less, 50 nm or less, 100 nm or less, 200 nm or less, 500nm or less, 800 nm or less, 1000 nm or less, 1 um or less, 2 um or less,3 um or less, 5 um or less, 10 um or less, 20 um or less, 30 um or less,50 um or less, 100 um or less, 150 um or less, 200 um or less, 300 um orless, 500 um or less, 800 um or less, 1 mm or less, 2 mm or less, 4 mmor less, or a range between any two of the values.

The spacer height and/or sample thickness is between 1 nm to 100 nm inone preferred embodiment, 100 nm to 500 nm in another preferredembodiment, 500 nm to 1,000 nm in a separate preferred embodiment, 1 um(i.e., 1,000 nm) to 2 um in another preferred embodiment, 2 um to 3 umin a separate preferred embodiment, 3 um to 5 um in another preferredembodiment, 5 um to 10 um in a separate preferred embodiment, and 10 umto 50 um in another preferred embodiment, 50 um to 100 um in a separatepreferred embodiment.

In some embodiments, the spacer height and/or sample thickness is (i)equal to or slightly larger than the minimum dimension of an analyte, or(ii) equal to or slightly larger than the maximum dimension of ananalyte. The “slightly larger” means that it is about 1% to 5% largerand any number between the two values.

In some embodiments, the spacer height and/or sample thickness is largerthan the minimum dimension of an analyte (e.g. an analyte has ananisotropic shape), but less than the maximum dimension of the analyte.

For example, the red blood cell has a disk shape with a minim dimensionof 2 um (disk thickness) and a maximum dimension of 11 um (a diskdiameter). In an embodiment of the present invention, the spacers isselected to make the inner surface spacing of the plates in a relevantarea to be 2 um (equal to the minimum dimension) in one embodiment, 2.2um in another embodiment, or 3 (50% larger than the minimum dimension)in other embodiment, but less than the maximum dimension of the redblood cell. Such embodiment has certain advantages in blood cellcounting. In one embodiment, for red blood cell counting, by making theinner surface spacing at 2 or 3 um and any number between the twovalues, a undiluted whole blood sample is confined in the spacing, onaverage, each red blood cell (RBC) does not overlap with others,allowing an accurate counting of the red blood cells visually. Too manyoverlaps between the RBC's can cause serious errors in counting.

In the present invention, in some embodiments, it uses the plates andthe spacers to regulate not only a thickness of a sample, but also theorientation and/or surface density of the analytes/entity in the samplewhen the plates are at the closed configuration. When the plates are ata closed configuration, a thinner thickness of the sample gives a lessthe analytes/entity per surface area (i.e. less surface concentration).

Spacer lateral dimension. For an open-spacer, the lateral dimensions canbe characterized by its lateral dimension (sometimes being called width)in the x and y—two orthogonal directions. The lateral dimension of aspacer in each direction is the same or different.

In some embodiments, the ratio of the lateral dimensions of x to ydirection is 1, 1.5, 2, 5, 10, 100, 500, 1,000, 10,000, or a rangebetween any two of the value. In some embodiments, a different ratio isused to regulate the sample flow direction; the larger the ratio, theflow is along one direction (larger size direction).

In some embodiments, the different lateral dimensions of the spacers inx and y direction are used as (a) using the spacers as scale-markers toindicate the orientation of the plates, (b) using the spacers to createmore sample flow in a preferred direction, or both.

In a preferred embodiment, the period, width, and height.

In some embodiments, all spacers have the same shape and dimensions. Insome embodiments, each spacer has different lateral dimensions.

For enclosed-spacers, in some embodiments, the inner lateral shape andsize are selected based on the total volume of a sample to be enclosedby the enclosed spacer(s), wherein the volume size has been described inthe present disclosure; and in certain embodiments, the outer lateralshape and size are selected based on the needed strength to support thepressure of the liquid against the spacer and the compress pressure thatpresses the plates.

Aspect ratio of height to the average lateral dimension of pillarspacer. In certain embodiments, the aspect ratio of the height to theaverage lateral dimension of the pillar spacer is 100,000, 10,000,1,000, 100, 10, 1, 0.1, 0.01, 0.001, 0.0001, 0, 00001, or a rangebetween any two of the values.

Spacer height precisions. The spacer height should be controlledprecisely. The relative precision of the spacer (i.e. the ratio of thedeviation to the desired spacer height) is 0.001% or less, 0.01% orless, 0.1% or less; 0.5% or less, 1% or less, 2% or less, 5% or less, 8%or less, 10% or less, 15% or less, 20% or less, 30% or less, 40% orless, 50% or less, 60% or less, 70% or less, 80% or less, 90% or less,99.9% or less, or a range between any of the values.

Inter-spacer distance. The spacers can be a single spacer or a pluralityof spacers on the plate or in a relevant area of the sample. In someembodiments, the spacers on the plates are configured and/or arranged inan array form, and the array is a periodic, non-periodic array orperiodic in some locations of the plate while non-periodic in otherlocations.

In some embodiments, the periodic array of the spacers has a lattice ofsquare, rectangle, triangle, hexagon, polygon, or any combinations ofthereof, where a combination means that different locations of a platehas different spacer lattices.

In some embodiments, the inter-spacer distance of a spacer array isperiodic (i.e. uniform inter-spacer distance) in at least one directionof the array. In some embodiments, the inter-spacer distance isconfigured to improve the uniformity between the plate spacing at aclosed configuration.

The distance between neighboring spacers (i.e. the inter-spacerdistance) is 1 um or less, 5 um or less, 10 um or less, 20 um or less,30 um or less, 40 um or less, 50 um or less, 60 um or less, 70 um orless, 80 um or less, 90 um or less, 100 um or less, 200 um or less, 300um or less, 400 um or less, or in a range between any two of the values.

In certain embodiments, the inter-spacer distance is at 400 or less, 500or less, 1 mm or less, 2 mm or less, 3 mm or less, 5 mm or less, 7 mm orless, 10 mm or less, or any range between the values. In certainembodiments, the inter-spacer distance is a 10 mm or less, 20 mm orless, 30 mm or less, 50 mm or less, 70 mm or less, 100 mm or less, orany range between the values.

The distance between neighboring spacers (i.e. the inter-spacerdistance) is selected so that for a given properties of the plates and asample, at the closed-configuration of the plates, the sample thicknessvariation between two neighboring spacers is, in some embodiments, atmost 0.5%, 1%, 5%, 10%, 20%, 30%, 50%, 80%, or any range between thevalues; or in certain embodiments, at most 80%, 100%, 200%, 400%, or arange between any two of the values.

Clearly, for maintaining a given sample thickness variation between twoneighboring spacers, when a more flexible plate is used, a closerinter-spacer distance is needed.

In a preferred embodiment, the spacer is a periodic square array,wherein the spacer is a pillar that has a height of 2 to 4 um, anaverage lateral dimension of from 5 to 20 um, and inter-spacer spacingof 1 um to 100 um.

In a preferred embodiment, the spacer is a periodic square array,wherein the spacer is a pillar that has a height of 2 to 4 um, anaverage lateral dimension of from 5 to 20 um, and inter-spacer spacingof 100 um to 250 um.

In a preferred embodiment, the spacer is a periodic square array,wherein the spacer is a pillar that has a height of 4 to 50 um, anaverage lateral dimension of from 5 to 20 um, and inter-spacer spacingof 1 um to 100 um.

In a preferred embodiment, the spacer is a periodic square array,wherein the spacer is a pillar that has a height of 4 to 50 um, anaverage lateral dimension of from 5 to 20 um, and inter-spacer spacingof 100 um to 250 um.

The period of spacer array is between 1 nm to 100 nm in one preferredembodiment, 100 nm to 500 nm in another preferred embodiment, 500 nm to1000 nm in a separate preferred embodiment, 1 um (i.e. 1000 nm) to 2 umin another preferred embodiment, 2 um to 3 um in a separate preferredembodiment, 3 um to 5 um in another preferred embodiment, um to 10 um ina separate preferred embodiment, and 10 um to 50 um in another preferredembodiment, 50 um to 100 um in a separate preferred embodiment, 100 umto 175 um in a separate preferred embodiment, and 175 um to 300 um in aseparate preferred embodiment.

Spacer density. The spacers are arranged on the respective plates at asurface density of greater than one per um², greater than one per 10um², greater than one per 100 um², greater than one per 500 um², greaterthan one per 1,000 um², greater than one per 5,000 um², greater than oneper 0.01 mm², greater than one per 0.1 mm², greater than one per 1 mm²,greater than one per 5 mm², greater than one per 10 mm², greater thanone per 100 mm², greater than one per 1000 mm², greater than one per10000 mm², or a range between any two of the values.

The spacers are configured to not take significant surface area (volume)in a given sample area (volume);

Ratio of spacer volume to sample volume. In many embodiments, the ratioof the spacer volume (i.e., the volume of the spacer) to sample volume(i.e. the volume of the sample), and/or the ratio of the volume of thespacers that are inside of the relevant volume of the sample to therelevant volume of the sample are controlled for achieving certainadvantages. The advantages include, but not limited to, the uniformityof the sample thickness control, the uniformity of analytes, the sampleflow properties (i.e., flow speed, flow direction, etc.).

In certain embodiments, the ratio of the spacer volume r) to samplevolume, and/or the ratio of the volume of the spacers that are inside ofthe relevant volume of the sample to the relevant volume of the sampleis less than 100%, at most 99%, at most 70%, at most 50%, at most 30%,at most 10%, at most 5%, at most 3% at most 1%, at most 0.1%, at most0.01%, at most 0.001%, or a range between any of the values.

Spacers fixed to plates. The inter spacer distance and the orientationof the spacers, which play a key role in the present invention, arepreferably maintained during the process of bringing the plates from anopen configuration to the closed configuration, and/or are preferablypredetermined before the process from an open configuration to a closedconfiguration.

Some embodiments of the present invention is that the spacers are fixedon one of the plates before the plates are brought to the closedconfiguration. The term “a spacer is fixed with its respective plate”means that the spacer is attached to a plate and the attachment ismaintained during a use of the plate. An example of “a spacer is fixedwith its respective plate” is that a spacer is monolithically made ofone piece of material of the plate, and the position of the spacerrelative to the plate surface does not change. An example of “a spaceris not fixed with its respective plate” is that a spacer is glued to aplate by an adhesive, but during a use of the plate, the adhesive cannothold the spacer at its original location on the plate surface (i.e. thespacer moves away from its original position on the plate surface).

In some embodiments, at least one of the spacers are fixed to itsrespective plate. In certain embodiments, at two spacers are fixed toits respective plates. In certain embodiments, a majority of the spacersare fixed with their respective plates. In certain embodiments, all ofthe spacers are fixed with their respective plates.

In some embodiments, a spacer is fixed to a plate monolithically.

In some embodiments, the spacers are fixed to its respective plate byone or any combination of the following methods and/or configurations:attached to, bonded to, fused to, imprinted, and etched.

The term “imprinted” means that a spacer and a plate are fixedmonolithically by imprinting (i.e. embossing) a piece of a material toform the spacer on the plate surface. The material can be single layerof a material or multiple layers of the material.

The term “etched” means that a spacer and a plate are fixedmonolithically by etching a piece of a material to form the spacer onthe plate surface. The material can be single layer of a material ormultiple layers of the material.

The term “fused to” means that a spacer and a plate are fixedmonolithically by attaching a spacer and a plate together, the originalmaterials for the spacer and the plate fused into each other, and thereis clear material boundary between the two materials after the fusion.

The term “bonded to” means that a spacer and a plate are fixedmonolithically by binding a spacer and a plate by adhesion.

The term “attached to” means that a spacer and a plate are connectedtogether.

In some embodiments, the spacers and the plate are made in the samematerials. In other embodiment, the spacers and the plate are made fromdifferent materials. In other embodiment, the spacer and the plate areformed in one piece. In other embodiment, the spacer has one end fixedto its respective plate, while the end is open for accommodatingdifferent configurations of the two plates.

In other embodiment, each of the spacers independently is at least oneof attached to, bonded to, fused to, imprinted in, and etched in therespective plate. The term “independently” means that one spacer isfixed with its respective plate by a same or a different method that isselected from the methods of attached to, bonded to, fused to, imprintedin, and etched in the respective plate.

In some embodiments, at least a distance between two spacers ispredetermined (“predetermined inter-spacer distance” means that thedistance is known when a user uses the plates).

In some embodiments of all methods and devices described herein, thereare additional spacers besides to the fixed spacers.

In one preferred embodiment, the spacers are monolithically made on thePlate by embossing (e.g. nanoimprinting) a thin plastic film using amold, and are made of the same materials, and the thickness of the Plateis from 50 um to 500 um.

In one preferred embodiment, the spacers are monolithically made on thePlate by embossing (e.g. nanoimprinting) a thin plastic film using amold, and are made of the same materials, and the thickness of the Plateis from 50 um to 250 um.

In one preferred embodiment, the spacers are monolithically made on thePlate and are made of the same materials, and the thickness of the Plateis from 50 um to 500 um.

In one preferred embodiment, the spacers are monolithically made on thePlate a thin plastic film using a mold, and are made of the samematerials, and the thickness of the Plate is from 50 um to 250 um.

In one preferred embodiment, the spacers are monolithically made on thePlate by embossing (e.g. nanoimprinting) a thin plastic film using amold, and are made of the same materials, where the plastic film areeither PMMA (polymethyl methacrylate) of PS (polystyrene).

In one preferred embodiment, the spacers are monolithically made on thePlate by embossing (e.g. nanoimprinting) a thin plastic film using amold, and are made of the same materials, where the plastic film areeither PMMA (polymethyl methacrylate) of PS (polystyrene) and thethickness of the Plate is from 50 um to 500 um.

In one preferred embodiment, the spacers are monolithically made on thePlate by embossing (e.g. nanoimprinting) a thin plastic film using amold, and are made of the same materials, where the plastic film areeither PMMA (polymethyl methacrylate) of PS (polystyrene) and thethickness of the Plate is from 50 um to 250 um.

In one preferred embodiment, the spacers are monolithically made on thePlate by embossing (e.g. nanoimprinting) a thin plastic film using amold, and are made of the same materials, where the plastic film areeither PMMA (polymethyl methacrylate) of PS (polystyrene), and thespacers have either a square or rectangle shape, and have the samespacer height.

In one preferred embodiment, the spacers have a square or rectangleshape (with or without round corners).

In one preferred embodiment, the spacers have square or rectanglepillars with the pillar width (spacer width in each lateral direction)between 1 um to 200 um; pillar period (i.e. spacer period) from 2um-2000 um, and pillar height (i.e. spacer height) from 1 um-100 um.

In one preferred embodiment, the spacers made of PMMA or PS have squareor rectangle pillars with the pillar width (spacer width in each lateraldirection) between 1 um to 200 um; pillar period (i.e. spacer period)from 2 um-2000 um, and pillar height (i.e. spacer height) from 1 um-100um.

In one preferred embodiment, the spacers are monolithically made on thePlate and are made of plastic materials, and the spacers have square orrectangle pillars with the pillar width (spacer width in each lateraldirection) between 1 um to 200 um; pillar period (i.e. spacer period)from 2 um-2,000 um, and pillar height (i.e. spacer height) from 1 um-100um.

In one preferred embodiment, the spacers are monolithically made on thePlate and are made of the same materials, and the spacers have square orrectangle pillars with the pillar width (spacer width in each lateraldirection) between 1 um to 200 um; pillar period (i.e. spacer period)from 2 um-2000 um, and pillar height (i.e. spacer height) from 1 um-10um.

In one preferred embodiment, the spacers are monolithically made on thePlate and are made of the same materials selected from PS or PMMA orother plastics, and the spacers have square or rectangle pillars withthe pillar width (spacer width in each lateral direction) between 1 umto 200 um; pillar period (i.e. spacer period) from 2 um-2,000 um, andpillar height (i.e. spacer height) from 10 um-50 um.

Specific sample thickness. In present invention, it was observed that alarger plate holding force (i.e. the force that holds the two platestogether) can be achieved by using a smaller plate spacing (for a givensample area), or a larger sample area (for a given plate-spacing), orboth.

In some embodiments, at least one of the plates is transparent in aregion encompassing the relevant area, each plate has an inner surfaceconfigured to contact the sample in the closed configuration; the innersurfaces of the plates are substantially parallel with each other, inthe closed configuration; the inner surfaces of the plates aresubstantially planar, except the locations that have the spacers; or anycombination of thereof.

Final Sample Thickness and Uniformity. In some embodiments,significantly flat is determined relative to the final sample thickness,and has, depending upon on embodiments and applications, a ratio of tothe sample thickness of less than 0.1%, less than 0.5%, less than 1%,less than 2%, less than 5%, or less than 10%, or a range between any twoof these values.

In some embodiments, flatness relative to the sample thickness can beless than 0.1%, less than 0.5%, less than 1%, less than 2%, less than5%, less than 10%, less than 20%, less than 50%, or less than 100%, or arange between any two of these values.

In some embodiments, significantly flat can mean that the surfaceflatness variation itself (measured from an average thickness) is lessthan 0.1%, less than 0.5%, less than 1%, less than 2%, less than 5%, orless than 10%, or a range between any two of these values. Generally,flatness relative to the plate thickness can be less than 0.1%, lessthan 0.5%, less than 1%, less than 2%, less than 5%, less than 10%, lessthan 20%, less than 50%, or less than 100%, or in a range between anytwo of these values.

The height of the spacers is selected by a desired regulated spacingbetween the plates and/or a regulated final sample thickness and theresidue sample thickness. The spacer height (the predetermined spacerheight), the spacing between the plates, and/or sample thickness is 3 nmor less, 10 nm or less, 50 nm or less, 100 nm or less, 200 nm or less,500 nm or less, 800 nm or less, 1000 nm or less, 1 μm or less, 2 μm orless, 3 μm or less, 5 μm or less, 10 μm or less, 20 μm or less, 30 μm orless, 50 μm or less, 100 μm or less, 150 μm or less, 200 μm or less, 300μm or less, 500 μm or less, 800 μm or less, 1 mm or less, 2 mm or less,4 mm or less, or in a range between any two of the values.

The spacer height, the spacing between the plates, and/or samplethickness is between 1 nm to 100 nm in one preferred embodiment, 100 nmto 500 nm in another preferred embodiment, 500 nm to 1,000 nm in aseparate preferred embodiment, 1 μm (i.e., 1,000 nm) to 2 μm in anotherpreferred embodiment, 2 μm to 3 μm in a separate preferred embodiment, 3μm to 5 μm in another preferred embodiment, 5 μm to 10 μm in a separatepreferred embodiment, and 10 μm to 50 μm in another preferredembodiment, 50 μm to 100 μm in a separate preferred embodiment.

In some embodiments, the spacers can be in spherical beads and randomlydistrusted in a sample.

In some embodiments, the QMAX device is fully transparent or partiallytransparent to reduce the heat absorption by card self, wherein thetransparence is above 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or arange between any two of the values.

In some embodiments, the QMAX device is partially reflective to reducethe heat absorption by card self. wherein the reflectance of the surfaceis above 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or in a range betweenany two of the values.

In some embodiments, the QMAX device and clamp is coated with a heatinsulator layer to reduce the heat absorption by card self. Wherein theheat insulator layer contains materials including the low thermalconductivity material above.

In some embodiments, the clamp covers and seals all the QMAX card inclose configuration.

In some embodiments, the clamp covers and seal only the perimeter of theQMAX card in close configuration.

In some embodiments, the clamp covers and seal only the perimeter of theQMAX card in close configuration, and not the heating and cooling zonearea.

In some embodiments, the clamp covers some of the surface of QMAX cardin close configuration.

In some embodiments, the clamp has a window which is transparent toallow the light go inside the QMAX card and out from the QMAX card.

In some embodiments, the clamp is fully transparent to allow the lightgo inside the QMAX card and out from the QMAX card.

wherein the transparence of the clamp is above 30%, 40%, 50%, 60%, 70%,80%, 90%, 100%, or a range between any two of the values.

In some embodiments, there is air or liquid between the clamp and QMAXdevice in close configuration. In certain embodiments, the liquidincludes but not limit to water, ethane, methane, oil, benzene, Hexane,heptane, silicone oil, polychlorinated biphenyls, liquid air, liquidoxygen, liquid nitrogen etc. In certain embodiments, the gas includesbut not limit to air, argon, helium, nitrogen, oxygen, carbon dioxide,etc.

In some embodiments, after close the clamp, the pressure on QMAX cardsurface applied by the clamp is 0.01 kg/cm², 0.1 kg/cm², 0.5 kg/cm², 1kg/cm², 2 kg/cm², kg/cm², 5 kg/cm², 10 kg/cm², 20 kg/cm², 30 kg/cm², 40kg/cm², 50 kg/cm², 60 kg/cm², 100 kg/cm², 150 kg/cm², 200 kg/cm², 500kg/cm², or a range between any two of the values; and a preferred rangeof 0.1 kg/cm² to 0.5 kg/cm², 0.5 kg/cm² to 1 kg/cm², 1 kg/cm² to 5kg/cm², 5 kg/cm² to 10 kg/cm² (Pressure).

In some embodiments, after close the clamp, the pressure on QMAX cardsurface applied by the clamp is at least 0.01 kg/cm², 0.1 kg/cm², 0.5kg/cm², 1 kg/cm², 2 kg/cm², kg/cm², 5 kg/cm², 10 kg/cm², 20 kg/cm², 30kg/cm², 40 kg/cm², 50 kg/cm², 60 kg/cm², 100 kg/cm², 150 kg/cm², 200kg/cm², or 500 kg/cm²,

As shown in the cross-sectional views of the device in FIG. 7A and FIG.7B, the heating/cooling layer 112 spans across the sample contact area.It should be noted, however, it is also possible that the lateral areaof the heating/cooling layer occupy only a portion of the sample contactarea at a percentage about 1% or more, 5% or more, 10% or more, 20% ormore, 50% or more, 80% or more, 90% or more, 95% or more, 99% or more,85% or less, 75% or less, 55% or less, 40% or less, 25% or less, 8% orless, 2.5% or less. In some embodiments, in order to facilitate thetemperature change of the sample, in some embodiments the lateral areaof the heating/cooling layer is configured so that the sample 90 receivethe thermal radiation from the heating/cooling layer 112 substantiallyuniformly across the lateral dimension of the sample 90 over the samplecontact area.

In some embodiments, the radiation absorbing area is 10%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 100% the total plate area, or a range between anytwo of the values.

In some embodiments, the heating/cooling layer 112 have a thickness of10 nm or more, 20 nm or more, 50 nm or more, 100 nm or more, 200 nm ormore, 500 nm or more, 1 um or more, 2 um or more, 5 um or more, 10 um ormore, 20 um or more, 50 um or more, 100 um or more, 75 um or less, 40 umor less, 15 um or less, 7.5 um or less, 4 um or less, 1.5 um or less,750 nm or less, 400 nm or less, 150 nm or less, 75 nm or less, 40 nm orless, or 15 nm or less, or in a range between any of the two values. Incertain embodiments, the heating/cooling layer 112 have thickness of 100nm or less.

In some embodiments, the area of the sample layer and theheating/cooling layer 112 is substantially larger than the uniformthickness. Here, the term “substantially larger” means that the generaldiameter or diagonal distance of the sample layer and/or theheating/cooling layer is at least 10 time, 15 times, 20 time, 25 times,30 time, 35 times, 40 time, 45 times, 50 time, 55 times, 60 time, 65times, 70 time, 75 times, 80 time, 85 times, 90 time, 95 times, 100time, 150 times, 200 time, 250 times, 300 time, 350 times, 400 time, 450times, 500 time, 550 times, 600 time, 650 times, 700 time, 750 times,800 time, 850 times, 900 time, 950 times, 1u000 time, 1,500 times, 2,000time, 2,500 times, 3,000 time, 3,500 times, 4,000 time, 4,500 times, or5000 time, or in a range between any of the two values.

In some embodiments, the heating/cooling layer has an area that is lessthan 1000 mm², 900 mm², 800 mm², 700 mm², 600 mm², 500 mm², 400 mm², 300mm², 200 mm², 100 mm², 90 mm², 80 mm², 75 mm², 70 mm², 60 mm², 50 mm²,40 mm², 30 mm², 25 mm², 20 mm², 10 mm², 5 mm², 2 mm², 1 mm², 0.5 mm²,0.2 mm², 0.1 mm², or 0.01 mm², or in a range between any of the twovalues. In some embodiments, the heating/cooling layer has an area thatis substantially smaller than the area of the first plate (and/or thesecond plate). For example, in certain embodiments, area of theheating/cooling layer occupy only a portion of the area of the firstplate (or the second plate; or the sample contact area of the firstplate or the second plate) at a percentage about 1% or more, 5% or more,10% or more, 20% or more, 50% or more, 80% or more, 90% or more, 95% ormore, 99% or more, 85% or less, 75% or less, 55% or less, 40% or less,25% or less, 8% or less, 2.5% or less.

In some embodiments, the heating/cooling layer has a substantiallyuniform thickness. In some embodiments, the heating/cooling layer has athickness of less than 10 nm, 20 nm, 50 nm, 100 nm, 200 nm, 500 nm, 1um, 2 um, 5 um, 10 um, 20 um, 50 um, 100 um, 200 um, 300 um, 400 um, 500um, 600 um, 700 um, 800 um, 900 um, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm,3.5 mm, 4 mm, 4.5 mm, 5 mm, or 10 mm, or in a range between any of thetwo values.

The heating/cooling layer can take any shape. For example, from a topview the heating/cooling layer can be square, circle, ellipse, triangle,rectangle, parallelogram, trapezoid, pentagon, hexagon, octagon,polygon, or various other shapes.

In some embodiments, the first plate or the second plate has a thicknessof 2 nm or less, 10 nm or less, 100 nm or less, 200 nm or less, 500 nmor less, 1,000 nm or less, 2 μm (micron) or less, 5 μm or less, 10 μm orless, 20 μm or less, 50 μm or less, 100 μm or less, 150 μm or less, 200μm or less, 300 μm or less, 500 μm or less, 800 μm or less, 1 mm(millimeter) or less, 2 mm or less, 3 mm or less, 5 mm or less, 10 mm orless, 20 mm or less, 50 mm or less, 100 mm or less, 500 mm or less, orin a range between any two of these values.

In some embodiments, the first plate and the second plate has a lateralarea of 1 mm² (square millimeter) or less, 10 mm² or less, 25 mm² orless, 50 mm² or less, 75 mm² or less, 1 cm² (square centimeter) or less,2 cm² or less, 3 cm² or less, 4 cm² or less, 5 cm² or less, 10 cm² orless, 100 cm² or less, 500 cm² or less, 1,000 cm² or less, 5,000 cm² orless, 10,000 cm² or less, 10,000 cm² or less, or in a range between anytwo of these values.

In certain embodiments, a fourth power of the inter-spacer-distance(ISD) of the spacers divided by the thickness (h) and the Young'smodulus (E) of the plate (ISD⁴/(hE)) is 5×10⁶ um³/GPa or less;

In certain embodiments, a product of the pillar contact filling factorand the Young's modulus of the spacers is 2 MPa or larger, wherein thepillar contact filling factor is the ratio of pillar contact area (thatcontact the plate at a closed configuration) inside a relevant samplevolume to the total plate area in the relevant sample volume.

In certain embodiments, the spacers have a predetermined substantiallyuniform height and a predetermined constant inter-spacer distance thatis at least about 2 times larger than the size of the analyte, up to 200um, and wherein at least one of the spacers is inside the sample contactarea.

In some embodiments, the plate (either the first plate, the secondplate, or both plates) that has the heating/cooling layer is thin sothat the temperature of the sample can be rapidly changed. For example,in certain embodiments the plate that is in contact with theheating/cooling layer has a thickness equal to or less than 500 um, 200um, 100 um, 50 um, 25 um, 10 um, 5 um, 2.5 um, 1 um, 500 nm, 400 nm, 300nm, 200 nm, or 100 nm, or in a range between any of the two values. Insome embodiments, if only one plate is on contact with theheating/cooling layer, the plate in contact with the heating/coolinglayer is substantially thinner than the plate that is not in contactwith the heating/cooling layer. For example, in some embodiments, thethickness of the plate that is in contact with the heating/cooling layeris less than 1/1,000,000, 1/500,000, 1/100,000, 1/50,000, 1/10,000,1/5,000, 1/1,000, 1/500, 1/100, 1/50, 1/10, ⅕, or ½ of the thickness ofthe plate that is in contact with the heating/cooling layer, or in arange between any of the two values.

In some embodiments, the sample layer is thin so that the temperature ofthe sample layer can be rapidly changed. In certain embodiments, thesample layer has a thickness equal to or less than 100 um, 50 um, 25 um,10 um, 5 um, 2.5 um, 1 um, 500 nm, 400 nm, 300 nm, 200 nm, or 100 nm, orin a range between any of the two values.

In various embodiments, the positioning of the heating/cooling layer canalso vary.

As herein shown and described, in some embodiments, the sample holder isconfigured to compress the fluidic sample into a thin layer, thusreducing the thermal mass of the sample. But reducing the thermal mass,a small amount energy can be able to change the temperature of thesample quickly. In addition, by limiting the sample thickness, thethermal conduction is also limited.

In some embodiments, there is a sample contact area on the respectivesurfaces of the first plate 10 and the second plate 20. The samplecontact area can be any portion of the surface of the first plate 10and/or the second plate 20. In some embodiments, the heating/coolinglayer at least partly overlaps with the sample contact area. In theoverlapping part, the sample is heated quickly due to close proximityand small thermal mass.

In some embodiments, the sample holder 100 is a compressed regulatedopen flow (CROF, also known as QMAX) device, such as but not limited tothe CROF device described in U.S. Provisional Patent Application No.62/202,989, which was filed on Aug. 10, 2015, U.S. Provisional PatentApplication No. 62/218,455, which was filed on Sep. 14, 2015, U.S.Provisional Patent Application No. 62/293,188, which was filed on Feb.9, 2016, U.S. Provisional Patent Application No. 62/305,123, which wasfiled on Mar. 8, 2016, U.S. Provisional Patent Application No.62/369,181, which was filed on Jul. 31, 2016, U.S. Provisional PatentApplication No. 62/394,753, which was filed on Sep. 15, 2016, PCTApplication (designating U.S.) No. PCT/US2016/046437, which was filed onAug. 10, 2016, PCT Application (designating U.S.) No. PCT/US2016/051775,which was filed on Sep. 14, 2016, PCT Application (designating U.S.) No.PCT/US2016/051794, which was filed on Sep. 15, 2016, and PCT Application(designating U.S.) No. PCT/US2016/054025, which was filed on Sep. 27,2016, the complete disclosures of which are hereby incorporated byreference for all purposes.

Edge Sealing for Reducing Sample Evaporation

When the two plates sandwich a sample into a shape with a large lateralto vertical ratio (e.g., 15 mm vs 30 um=500), the evaporation of thesample during a thermal cycling is greatly reduced, since the samplesurfaces covered by the two plate is 500 times larger. Experimentally,we found that in 30 temperature cycling (about 60 secs), there was novisible changes in the sample volume.

On the other hand, in some embodiments, it has a seal element that is incontact with the two plates to form an enclosed chamber which preventssample vapor going out. Such seal element can reduce samplecontamination, in addition to reduce or eliminate sample evaporation.The sealing element can be a tape, plastic seal, oil seal, or acombination of thereof.

In some embodiments, the sealing element does not reach the sample, butthe sealing element is in contact with the two plates to form anenclosed chamber which prevents sample vapor going out. In someembodiments, the sealing element can be used as spacers to regulate therelevant sample's thickness.

In some embodiments, as shown in FIG. 10 , the sample holder 100comprises a sealing element 30 that is configured to seal the spacing102 between the first plate 10 and second plate 20 outside the mediumcontact area at the closed configuration. In certain embodiments, thesealing element 30 encloses the sample 90 within a certain area (e.g.the sample receiving area) so that the overall lateral area of thesample 90 is well defined and measurable. In certain embodiments, thesealing element 30 improves the uniformity of the sample 90, especiallythe thickness of the sample layer.

In some embodiments, as shown in FIG. 10 , the sealing element 30comprises an adhesive applied between the first plate 10 and secondplate 20 at the closed configuration. The adhesive is selective frommaterials such as but not limited to: starch, dextrin, gelatin, asphalt,bitumen, polyisoprene natural rubber, resin, shellac, cellulose and itsderivatives, vinyl derivatives, acrylic derivatives, reactive acrylicbases, polychloroprene, styrene-butadiene, styrene-diene-styrene,polyisobutylene, acrylonitrile-butadiene, polyurethane, polysulfide,silicone, aldehyde condensation resins, epoxide resins, amine baseresins, polyester resins, polyolefin polymers, soluble silicates,phosphate cements, or any other adhesive material, or any combinationthereof. In some embodiments, the adhesive is drying adhesive,pressure-sensitive adhesive, contact adhesive, hot adhesive, or one-partor multi-part reactive adhesive, or any combination thereof. In someembodiments, the glue is natural adhesive or synthetic adhesive, or fromany other origin, or any combination thereof. In some embodiments, theadhesive is spontaneous-cured, heat-cured, UV-cured, or cured by anyother treatment, or any combination thereof.

In some embodiments, as shown in FIG. 10 , the sealing element 30comprises an enclosed spacer (well). For example, the enclosed spacerhas a circular shape (or any other enclosed shape) from a top view andencircle the sample 90, essentially restricting the sample 90 togetherwith the first plate 10 and the second plate 20. In certain embodiments,the enclosed spacer (well) also function as the spacing mechanism 40. Insuch embodiments, the enclosed spacer seals the lateral boundary of thesample 90 as well as regulate the thickness of the sample layer.

In some embodiments, there is an “evaporation-prevention ring” outsideof the liquid area (e.g. sample area) that prevents or reduces the vaporof the liquid escape the card, during a heating.

In some embodiments, there is a clamp outside of the QMAX-card to fixthe QMAX card in its closed configuration during a heating.

In some embodiments, the two plates are compressed by an imprecisepressing force, which is neither set to a precise level norsubstantially uniform. In certain embodiments, the two plates arepressed directly by a human hand.

In some embodiments, the QMAX card/RHC card, including the plates andspacer, is made of the material with low thermal conductivity to reducethe heat absorption by card self.

In some embodiments, there is clamp outside of the QMAX-card to fix theQMAX card in its closed configuration during a heating (namely, theclamp clamps only round the edge of the plates, not the center of theplate pair). In some embodiments, the clamp is made of the material withlow thermal conductivity to reduce the heat absorption by card self.

Heating Source, Extra Heat Sink, Temperature Sensor, and TemperatureControl

The heating layer or the heating/cooling layer in a RHC card isconfigured to be heated by a heating source, wherein the heating sourcedelivers heat energy to the heating/cooling layer optically,electrically, by radio frequency (RF) radiation, or a combinationthereof.

Optical Heating Source. In some embodiments, when a heating layer isheated by a heating source optically, the heating source comprises alight source, that include, but not limited to, LED (light emittingdiode), lasers, lamps, or a combination of thereof.

To get more light from a light source in an optical heating source to aheating layer, some embodiments of the heating sources uses an opticallens, an optical pipe, or a combination thereof.

In some embodiments, the wavelength of the electromagnetic waves is 50nm, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500nm, 550 nm, 600 nm, 650 nm, 700 nm, 750 nm, 800 nm, 850 nm, 900 nm, 950nm, 1 um, 10 um, 25 um, 50 um, 75 um, or 100 um, or in a range betweenany of the two values. In some embodiments, the wavelength of theelectromagnetic waves is 100 nm to 300 nm, 400 nm to 700 nm (visiblerange), 700 nm to 1000 nm (IR range), 1 um to 10 um, 10 um to 100 um, orin a range between any of the two values.

The lens has an NA (numerical aperture) of 0.001, 0.01, 0.05, 0.1, 0.2,0.3, 04, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.5, or in a range betweenany of the two values.

In preferred embodiments, the lens has an NA in a range of 0.01 to 0.1,0.1 to 0.4, 0.4 to 0.7, 0.7 to 1.0, or 1.0 to 1.5.

Electrical Heating Source. In some embodiments, when the heating layeror the heating/cooling layer is heated by a heating source electrically,the electric heating source comprises an electrical power supply thatsends an electrical power, though electrical wiring, to theheating/cooling layer.Extra Heat Sink. In some embodiments, the heat is removed from thesample and the sample holder to the environment, but in someembodiments, extra heat sink will be used to accelerate the heatremoval. The extra heat sink can be a Peltier cooler, passive heatradiator, or both. In some embodiments, fan will be used to create airconvention (directly to the sample and the sample holder, directly toextra heat sink, or both) which accelerate a cooling of the sample.Temperature sensors. The temperature of the sample can be controlled bydelivering pre-calibrated energy to the heating zone/layer with a realtime temperature sensor, by using a real time temperature sensor, orboth.

A real time temperature sensor can be thermometer, thermal couple,radiation temperature sensor, temperature sensitive dye (which changeeither light intensity or color or both with temperature), or acombination thereof.

As shown in FIG. 10 , in some embodiments the thermal control unit 200comprises a thermometer 206. In some embodiments, the thermometer 206provides a monitoring and/or feedback mechanism tocontrol/monitor/adjust the temperature of the sample 90. For example, insome embodiments the thermometer 206 is configured to measure thetemperature at or in proximity of the sample contact area. In certainembodiments, the thermometer 206 is configured to directly measure thetemperature of the sample 90. In some embodiments, the thermometer 206is selected from the group consisting of: fiber optical thermometer,infrared thermometer, fluidic crystal thermometer, pyrometer, quartzthermometer, silicon bandgap temperature sensor, temperature strip,thermistor, and thermocouple. In certain embodiments, the thermometer206 is an infrared thermometer.

In some embodiments, the thermometer 206 is configured to send signalsto the controller 204. Such signals comprise information related to thetemperature of the sample 90 so that the controller 204 makescorresponding changes. For example, during a PCR, for the denaturationstep the target temperature is set for 95° C.; after measurement, thethermometer sends a signal to the controller 204, indicating that themeasured temperature of the sample 90 is actually 94.8° C.; thecontroller 204 thus alters the output the heating source 202, whichprojects an electromagnetic wave or adjust particular parameters (e.g.,intensity or frequency) of an existing electromagnetic wave so that thetemperature of the sample 90 is increased to 95° C. Suchmeasurement-signaling-adjustment loop is applied to any step in anyreaction/assay.

Controllers. Referring to panels (A) and (B) of FIG. 9 , the controller204 is configured to control the electromagnetic wave 210 projected fromthe heating source 202 for the temperature change of the sample. Theparameters of the electromagnetic wave 210 that the controller 204controls include, but are not limited to, the presence, intensity,wavelength, incident angle, and any combination thereof. In someembodiments, the controller is operated manually, for instance, it is assimple as a manual switch that controls the on and off of the heatingsource, and therefore the presence of the electromagnetic wave projectedfrom the heating source. In other embodiments, the controller includeshardware and software that are configured to control the electromagneticwave automatically according to one or a plurality of pre-determinedprograms.

In some embodiments, the pre-determined program refers to a schedule inwhich the parameter(s) (e.g., presence, intensity, and/or wavelength) ofthe electromagnetic wave 210 is/are set to pre-determined levels forrespective pre-determined periods of time. In other embodiments, thepre-determined program refers to a schedule in which the temperature ofthe sample 90 is set to pre-determined levels for respectivepre-determined periods of time and the time periods for the change ofthe sample temperature from one pre-determined level to anotherpre-determined level are also set respectively. In some embodiments, thecontroller 204 is configured to be programmable, which means thecontroller 204 comprises hardware and software that are configured toreceive and carry out pre-determined programs for the system that aredelivered by the operator of the system.

FIG. 10 shows a sectional view of an embodiment of the presentinvention, demonstrating the thermal cycler system and showingadditional elements that facilitates temperature change and control. Asshown in FIG. 10 , the thermal cycler system comprises a sample holder100 and a thermal control unit 200. The sample holder 100 comprises afirst plate 10, a second plate 20, a spacing mechanism 40, and a sealingelement 30; the thermal control unit 200 comprises a heating source 202,a controller 204, a thermometer 206, and an expander 208.

FIG. 10 shows the sample holder 100 in a closed configuration, in whichthe inner surfaces 11 and 21 of the first and second plates 10 and 20face each other and the spacing 102 between the two plates are regulatedby a spacing mechanism 40. If a sample 90 has been deposited on one orboth of the plates in the open configuration, when switching to theclosed configuration, the first plate 10 and the second plate 20 arepressed by a human hand or other mechanisms, the sample 90 is thuscompressed by the two plates into a thin layer. In some embodiments, thethickness of the layer is uniform and the same as the spacing 102between the two plates. In certain embodiments, the spacing 102 (andthus the thickness of the sample layer) is regulated by the spacingmechanism 40. In some embodiments, the spacing mechanism comprises anenclosed spacer that is fixed to one of the plates. In some embodiments,the spacing mechanism 40 comprises a plurality of pillar shaped spacersthat are fixed to one or both of the plates. Here the term “fixed” meansthat the spacer(s) is attached to a plate and the attachment ismaintained during at least a use of the plate.

In some embodiments, the controller 204 is configured to adjust thetemperature of the sample to facilitate an assay and/or reactioninvolving the sample 90 according to a pre-determined program. In someembodiments, the assay and/or reaction is a PCR. In certain embodiments,the controller 204 is configured to control the presence, intensity,and/or frequency of the electromagnetic wave from the heating source206.

Sample Signal Monitoring

As shown in FIGS. 11 and 12 , a signal sensor can be used to detect thesignal from the sample (and the products from a reaction during atemperature change) in the sample holder.

In some embodiments, the signal sensor is an optical sensor that isconfigured to image the fluidic sample. For example, optical sensor is aphotodetector, camera, or a device capable of capturing images of thefluidic sample. In some embodiments, the optical sensor can be a camera.In some embodiments, the camera is a camera integrated into a mobiledevice (e.g. a smartphone or tablet computer). In some embodiments, thecamera is separated from other parts of the system. In some embodiments,a light source or multi light sources are used to excite the sample (andthe products from a reaction during a temperature change) for generatinga signal

In some embodiments, the signal sensor is an electrical sensor that isconfigured to detect electrical signals from the device. In someembodiments, the signal sensor is a mechanical sensor that is configuredto detect mechanical signals from the device.

In some embodiments, the signal sensor is configured to monitor theamount of an analyte in the sample. In some embodiments, the signalsensor is outside the chamber and receive optical signals from thesample through an optical aperture on the chamber.

Base and Systems

In some embodiments, the apparatus further comprises a base (an adaptor)that is configured to house the sample card, the heating source,temperature sensors, a part of an entire of temperature controlled(include a smartphone in some embodiments), extra-heat sink(optionally), a fan (optionally) or a combination of thereof. In someembodiments, the adaptor comprises a card slot, into which the samplecard can be inserted. In some embodiments, the sample card, after beingfully inserted into the slot, or after reaching a pre-defined positionin the slot, is stabilized and stays in place without any movement.

In some embodiments, the base (adaptor) is configured to position thesample card, and the sample within the sample card, in the field of viewof an optical sensor (e.g. a camera) so that the sample can be imaged.In certain embodiments, the camera is part of a mobile device (e.g. asmartphone). In some embodiments, the adaptor comprises a slider in theslot. In certain embodiments, the sample card can be put onto theslider, which can slide into or out of the slot in the adaptor. In someembodiments, the adaptor comprises a card support. In certainembodiments, the sample card can be put on the card support, which doesnot need to be moved before imaging.

In some embodiments, the adaptor is configured to be connectable to anoptical sensor so that the relative position of the optical sensor(e.g., mobile device; e.g., smartphone) and the sample card is fixed. Incertain embodiments, the adaptor can include a connecting member that isreplaceable and directly attach to the mobile device (as an example).The connecting member can be slid onto the mobile device and firmlyattach the adaptor to the mobile device, optimally positioning thesample card to be imaged or for the detection and/or measurement of theanalyte. In certain embodiments, the connecting member is replaceable sothat different connecting members can be used for different mobiledevices.

In some embodiments, the adaptor comprises a radiation aperture thatallows the passage of the electromagnetic waves that heat or cool thesample. In some embodiments, the adaptor comprises an optical aperturethat allows imaging of the sample. In some embodiments, the adaptorserves as a heating sink for the sample card

As shown in FIG. 10 , the thermal control unit 200 comprises a beamexpander 208, which is configured to expand the electromagnetic wavefrom the heating source 202 from a smaller diameter to a largerdiameter. In some embodiments, the electromagnetic wave projected fromthe heating source 202 is sufficient to cover the entire sample contactarea; in some embodiments however, it is necessary to expand the coveredarea of the electromagnetic wave projected directed from the heatingsource 202 to produce an expanded electromagnetic wave 210, providing aheat source for all the sample contact area(s). The beam expander 208employs any known technology, including but not limited to the beamexpanders described in U.S. Pat. Nos. 4,545,677, 4,214,813, 4,127,828,and 4,016,504, and U.S. Pat. Pub. No. 2008/0297912 and 2010/0214659,which are incorporated by reference in their entireties for allpurposes.

Smartphone

In some embodiments, the sample card is imaged by a mobile device. Incertain embodiments, the mobile device is a smartphone, which can serveas an example.

In some embodiments, the smartphone comprises a camera that can be usedto image the sample in the sample card. In some embodiments, an adaptoris used to accommodate the sample card and the adaptor is configured toattach to the smartphone so that the sample card (and the sampletherein) can be placed in the field of view of the camera.

In some embodiments, the smartphone can also serve as the control unit,which is configured to control the apparatus. For example, thesmartphone can be used control the heating and/or cooling of the samplecard. In certain embodiments, the smartphone is connected to the heatingsource and controls the electromagnetic waves from the heating source.In some embodiments, the smartphone controls the presence, intensity,wavelength, frequency, and/or angle of the electromagnetic waves. Incertain embodiments, the smartphone receives the temperature data from athermometer that measures the temperature of the sample. In certainembodiments, the smartphone controls the electromagnetic waves based onthe temperature data.

In some embodiments, the smartphone can also serve as a data processingand communication device. For example, after the sample has been imaged,the images can be saved in the smart phone. In certain embodiments, thesave images can be processed by software or applications in thesmartphone. For example, the presence and/or amount of the analyte canbe deduced from the images by software or applications in thesmartphone. In certain embodiments, the processed results can bedisplayed on the screen of the smart phone. In certain embodiments, theprocessed results can be sent to the user, e.g. with email or othermessaging software. In certain embodiments, the processed results can besent to a third party, e.g., a healthcare professional, who can makefurther diagnostics and/or process the data in additional steps. In someembodiments, the images, without processing, can be displayed and/ortransmitted. In certain embodiments, the images are displayed on thescreen of the smartphone. In certain embodiments, the images are sent tothe user, e.g. by email or other messaging software. In certainembodiments, the images can be sent to a third party, e.g. a remoteserver, which can process the images further. In some embodiments, theresults and/or images are compressed and/or encrypted before being sent.

Use of RHC Card

The RHC card in the description can be used as one step of multiplesteps in test a sample, or as one step that perform entire test.

In some embodiments, a RHC card is used in a so-called “one-step assay”,wherein all reagents and a sample for an analysis are loaded on a RHCcard and a thermal cycling or temperature change is performed and thesignal is being observed during the thermal cycling or temperaturechange.

OTHER EMBODIMENTS Embodiment 1

One embodiment comprises a device of the embodiment SH-1 to SH-6,wherein the first plate and the second plate are flexible plastic filmand/or thin glass film, that each has a substantially uniform thicknessof a value selected from a range between 1 um to 25 um.

Each plate has an area in a range of 1 cm² to 16 cm².

The sample sandwiched between the two plate has a thickness of 40 um orless.

The relevant sample to the entire sample ratio (RE ratio) is 12% orless.

The cooling zone is at least 9 times larger than the heating zone.

The sample to non-sample thermal mass ratio is 2.2 or lager.

The RHC have no spacer in some embodiments, but do have spacers in otherembodiments.

STC ratio is and the cooling zone comprises a layer of the material thathas a thermal conductivity of 70 W/m-K or higher and a thermalconductivity times its thickness.

Embodiment 2

For the embodiments of SH-1 to SH-x, they have the following parameterarrange for fast thermal cycling.

The first plate and second plates are plastic or a thin glass. The firstplate and second plate have a thickness of 100 nm, 500 nm, 1 um, 5 um,10 um, in a range between any of the two values.

The sample between the two plates has a thickness of 5 um, 10 um, 30 um,50 um, 100 um, or in a range between any of the two values.

The distance from the H/C layer to the sample is 10 nm, 100 nm, 500 nm,1 um, 5 um, 10 um, or in a range between any of the two values.

The ratio of the cooling zone area to the relevant sample area is 16, 9,4, 2, or in a range between any of the two values.

The ratio of the cooling zone area to the heating area is 16, 9, 4, 2,or in a range between any of the two values.

The distance between the H/C layer and the heating source (e.g. LED) is5 mm, 10 mm, 20 mm, 30 mm, or in a range between any of the two values.

Embodiment 3

For the embodiments of SH-1 to SH-x, they have the following parameterarrange for fast thermal cycling.

The first plate and second plates are plastic or a thin glass. The firstplate has a thickness of 10 um, 25 um, 50 um, or in a range between anyof the two values; while the second plate (that plate that has heatinglayer or cooling layer) has a thickness of 100 nm, 500 nm, 1 um, 5 um,10 um, in a range between any of the two values.

The sample between the two plates has a thickness of 5 um, 10 um, 30 um,50 um, 100 um, or in a range between any of the two values.

The distance between the H/C layer and the sample is 10 nm, 100 nm, 500nm, 1 um, 5 um, 10 um, or in a range between any of the two values.

The ratio of the cooling zone area to the relevant sample area is 16, 9,4, 2, or in a range between any of the two values.

The ratio of the cooling zone area to the heating area is 16, 9, 4, 2,or in a range between any of the two values.

The distance between the H/C layer and the heating source (e.g. LED) is5 mm, 10 mm, 20 mm, 30 mm, or in a range between any of the two values.

Embodiment 4

For the embodiments of SH-1 to SH-x, they have the following parameterarrange for fast thermal cycling.

The first plate and second plates are plastic or a thin glass. The firstplate and second plate have a thickness of 100 nm, 500 nm, 1 um, 5 um,10 um, 25 um, 50 um, 100 um, 175 um, 250 um, or in a range between anyof the two values.

The sample between the two plates has a thickness of 100 nm, 500 nm, 1um, 5 um, 10 um, 25 um, 50 um, 100 um, 250 um, or in a range between anyof the two values.

The distance between the H/C layer and the sample is 100 nm, 500 nm, 1um, 5 um, 10 um, 25 um, 50 um, 100 um, 175 um, 250 um, or in a rangebetween any of the two values.

The ratio of the cooling zone area to the relevant sample area is 100,64, 16, 9, 4, 2, 1, 0.5, 0.1, or in a range between any of the twovalues.

The ratio of the cooling zone area to the heating zone is 100, 64, 16,9, 4, 2, 1, 0.5, 0.1, or in a range between any of the two values.

The distance between the H/C layer and the heating source (e.g. LED) is500 um, 1 mm, 3 mm, 5 mm, 10 mm, 20 mm, 30 mm, or in a range between anyof the two values.

Embodiment 5

For the embodiments of SH-1 to SH-5, they have the following parameterarrange for fast thermal cycling.

A light pipe collimates the light from a light source (e.g. LED) intothe heating zone. The light pile comprises a structure with a hollowhole (e.g. a tube or a structure milled a hole) with a reflective wall.The light pile has a lateral dimension for 1 mm to 8 mm and length of 2mm to 5o mm.

Embodiment 6

For the embodiments of SH-1 to SH-5, they have the following parameterarrange for fast thermal cycling.

The first plate and second plates are plastic or a thin glass. The firstplate and second plate have a thickness of 100 nm, 500 nm, 1 um, 5 um,10 um, in a range between any of the two values.

The sample between the two plates has a thickness in a range of 1 to 5um, 5 um to um, 10 to 30 um, or 30 um to 50 um.

The distance from the H/C layer to the sample is in a range of 10 nm to100 nm, 100 nm to 500 nm, 500 nm to 1 um, 1 um to 5 um, 5 um to 10 um,or 10 um to 25 um. The ratio of the cooling zone area to the relevantsample area is 16, 9, 4, 2, or in a range between any of the two values.

The ratio of the cooling zone area to the heating area is 16, 9, 4, 2,or in a range between any of the two values.

The distance between the H/C layer and the heating source (e.g. LED) is5 mm, 10 mm, 20 mm, 30 mm, or in a range between any of the two values.

The KC ratio for the cooling layer is in a range of between 0.5 cm²/secand 0.7 cm²/sec, 0.7 cm²/sec and 0.9 cm²/sec, 0.9 cm²/sec and 1 cm²/sec,1 cm²/sec and 1.1 cm²/sec, 1.1 cm²/sec and 1.3 cm²/sec, 1.3 cm²/sec and1.6 cm²/sec, 1.6 cm²/sec and 2 cm²/sec, or 2 cm²/sec and cm²/sec.

The sample to non-sample thermal mass ratio is in a range of between 0.2to 0.5, 0.5 to 0.7, 0.7 to 1, 1 to 1.5, 1.5 to 5, 5 to 10, 10 to 30, 30to 50, or 50 to 100.

Embodiment 7

For the embodiments of SH-1 to SH-5, as well as Embodiments 1 toEmbodiments 6, they have the following parameter arrange for fastthermal cycling:

The first plate and/or the second plate has a lateral area in a range of1 mm² (square millimeter) to 10 mm², 10 mm² to 50 mm², 50 mm² to 100mm², 1 cm² to 5 cm², 5 cm² to 20 cm², or 20 cm² to 50 cm².

The scaled thermal conduction ratio (STM ratio) is in a range of between10 to 20, 30 to 50, 50 to 70, 70 to 100, 100 to 1,000, 1,000 to 10,000,or 10,000 to 1,000,000; and the cooling zone (layer) has thermalconductivity times its thickness of 6×10⁻⁵ W/K, 9×10⁻⁵ W/K, 1.2×10⁻⁴W/K, 1.5×10⁻⁴ W/K, 1.8×10⁻⁴ W/K, 2.1×10⁻⁴ W/K, 2.7×10⁻⁴ W/K, 3×10⁻⁴ W/K,1.5×10⁻⁴ W/K, or in a range between any of the two values.

The sample holder (RHC card) has not significant thermal conduction tothe environment during a thermal cycling.

Sample Types

The devices, systems, and methods herein disclosed can be used forsamples such as but not limited to diagnostic sample, clinical sample,environmental sample and foodstuff sample. The types of sample includebut are not limited to the samples listed, described and summarized inPCT Application (designating U.S.) Nos. PCT/US2016/046437 andPCT/US2016/051775, which were respectively filed on Aug. 10, 2016 andSep. 14, 2016, and are hereby incorporated by reference by theirentireties.

For example, in some embodiments, the devices, systems, and methodsherein disclosed are used for a sample that includes cells, tissues,bodily fluids and/or a mixture thereof. In some embodiments, the samplecomprises a human body fluid. In some embodiments, the sample comprisesat least one of cells, tissues, bodily fluids, stool, amniotic fluid,aqueous humour, vitreous humour, blood, whole blood, fractionated blood,plasma, serum, breast milk, cerebrospinal fluid, cerumen, chyle, chime,endolymph, perilymph, feces, gastric acid, gastric juice, lymph, mucus,nasal drainage, phlegm, pericardial fluid, peritoneal fluid, pleuralfluid, pus, rheum, saliva, sebum, semen, sputum, sweat, synovial fluid,tears, vomit, urine, and exhaled breath condensate.

In some embodiments, the devices, systems, and methods herein disclosedare used for an environmental sample that is obtained from any suitablesource, such as but not limited to: river, lake, pond, ocean, glaciers,icebergs, rain, snow, sewage, reservoirs, tap water, drinking water,etc.; solid samples from soil, compost, sand, rocks, concrete, wood,brick, sewage, etc.; and gaseous samples from the air, underwater heatvents, industrial exhaust, vehicular exhaust, etc. In certainembodiments, the environmental sample is fresh from the source; incertain embodiments, the environmental sample is processed. For example,samples that are not in liquid form are converted to liquid form beforethe subject devices, systems, and methods are applied.

In some embodiments, the devices, systems, and methods herein disclosedare used for a foodstuff sample, which is suitable or has the potentialto become suitable for animal consumption, e.g., human consumption. Insome embodiments, a foodstuff sample includes raw ingredients, cooked orprocessed food, plant and animal sources of food, preprocessed food aswell as partially or fully processed food, etc. In certain embodiments,samples that are not in liquid form are converted to liquid form beforethe subject devices, systems, and methods are applied.

The subject devices, systems, and methods can be used to analyze anyvolume of the sample. Examples of the volumes include, but are notlimited to, about 10 mL or less, 5 mL or less, 3 mL or less, 1microliter (μL, also “uL” herein) or less, 500 μL or less, 300 μL orless, 250 μL or less, 200 μL or less, 170 μL or less, 150 μL or less,125 μL or less, 100 μL or less, 75 μL or less, 50 μL or less, 25 μL orless, 20 μL or less, 15 μL or less, 10 μL or less, 5 μL or less, 3 μL orless, 1 μL or less, 0.5 μL or less, 0.1 μL or less, 0.05 μL or less,0.001 μL or less, 0.0005 μL or less, 0.0001 μL or less, 10 μL or less, 1μL or less, or a range between any two of the values.

In some embodiments, the volume of the sample includes, but is notlimited to, about 100 μL or less, 75 μL or less, 50 μL or less, 25 μL orless, 20 μL or less, 15 μL or less, 10 μL or less, 5 μL or less, 3 μL orless, 1 μL or less, 0.5 μL or less, 0.1 μL or less, 0.05 μL or less,0.001 μL or less, 0.0005 μL or less, 0.0001 μL or less, 10 μL or less, 1pL or less, or a range between any two of the values. In someembodiments, the volume of the sample includes, but is not limited to,about 10 μL or less, 5 μL or less, 3 μL or less, 1 μL or less, 0.5 μL orless, 0.1 μL or less, 0.05 μL or less, 0.001 μL or less, 0.0005 μL orless, 0.0001 μL or less, 10 pL or less, 1 pL or less, or a range betweenany two of the values.

In some embodiments, the amount of the sample is about a drop of liquid.In certain embodiments, the amount of sample is the amount collectedfrom a pricked finger or fingerstick. In certain embodiments, the amountof sample is the amount collected from a microneedle, micropipette or avenous draw.

In certain embodiments, the sample holder is configured to hold afluidic sample. In certain embodiments, the sample holder is configuredto compress at least part of the fluidic sample into a thin layer. Incertain embodiments, the sample holder comprises structures that areconfigured to heat and/or cool the sample. In certain embodiments, theheating source provides electromagnetic waves that can be absorbed bycertain structures in the sample holder to change the temperature of thesample. In certain embodiments, the signal sensor is configured todetect and/or measure a signal from the sample. In certain embodiments,the signal sensor is configured to detect and/or measure an analyte inthe sample. In certain embodiments, the heat sink is configured toabsorb heat from the sample holder and/or the heating source. In certainembodiments, the heat sink comprises a chamber that at least partlyenclose the sample holder.

D. Imager Based Rapid Temperature Assaying and Real Time PCR

Imaging Based Temperature Sensor

In certain embodiments, during a thermal cycling process, one or moretemperature sensing images are used to monitoring the temperature of asample. The temperature sensing image can sense a local temperature atdifferent locations of a sample. One can determine a suitable heatingtemperature to the sample (e.g. control the heating power), based on thetemperature map of a sample, rather than just a single lump-sumtemperature.

When heating up the assay device, some air bubbles and other defectswill form and be trapped in the heating area of the assay device. Andthe temperature of sample liquid and air are different. If using alump-sum temperature sensor to measure the average temperature in theheating area to be used as the temperature of sample liquid, it is notaccurate. In order to accurately measure the temperature of the sampleliquid in the heating area, an image-based temperature sensor should beused in the system to distinguish the temperature between air bubble andsample liquid.

Some enbedments have a system of heating and temperature monitoringdevice for the assay device. A heating source is put under the assaydevice to heat up the assay device. And on top of the thermal imager,there is a thermal temperature sensor to monitor the temperature of theheating area on the assay device. The temperature sensor is a thermalimager whose field of view is aligned with the heating area.

Some enbedments have a thermal imager in the system described above whenheating up the assay device. For example, B1 and B2 are two air bubblesand/or defects which are generated and trapped in the assay deviceduring heating up. And S3 is the sample liquid region. Using the imagebased thermal sensor, we can tell the difference between the temperatureof the sample liquid T3 and the air bubble region temperatures T1 andT2. So that we can get more accurate temperature of the sample liquid inthe heating area.

In this experiment, the assay device has a top (first) PMMA plate with50 um thickness, pillar array with 30 um pillar height, 30 um by 40 umpillar size, and 80 um inter pillar distance; a bottom (second) PETplate with 50 um thickness. A heating/cooling layer is on the outersurface of the second plate, and covers the entire second plate outersurface. The heating/cooling layer comprises an Au (gold) film and ablack paint layer. The gold film has one surface in contact with thesecond plate outer surface, and another surface being painted with ablack paint. The black paint is a commercial product of a filmcomposited of black carbon nanoparticle and polymer mixture. The blackpaint had an average thickness of ˜9 um (˜2 um thickness variation). Theblack paint layer may be directly facing incoming LED light. Between theAu film and the second plate outer surface, there is a 5 nm adhesionlayer of Ti, which improves the adhesion between Au and the secondplate. The heating source is a blue light emitting diode (LED) with acentral wavelength of 450 nm and power consumption around 500 mW.

-   -   1. In certain embodiments, a system, comprising:        -   (i) a device, comprising:        -   a first plate comprising a polymer material and having a            thickness less than or equal to 100 μm,        -   a second plate comprising a polymer material and having a            thickness less than or equal to 100 μm, wherein the second            plate is separated from the first plate in a parallel            arrangement by a distance less than or equal to the            thickness of the second plate,        -   a heating/cooling layer disposed on either the first plate            or the second plate, the heating/cooling layer having a            thickness and a thermal conductivity between 6×10⁻⁵ W/K            multiplied by the thickness of the heating/cooling layer and            1.5×10⁻⁴ W/K multiplied by the thickness of the            heating/cooling layer, and        -   a support frame configured to support at least one of the            first plate and the second plate;        -   an optical source configured to direct electromagnetic            radiation towards the heating/cooling layer,        -   a temperature sensor to monitor the temperature of the            heating area in the device;        -   wherein the heating/cooling layer is configured to absorb at            least a portion of the electromagnetic radiation such that            at least a portion of a liquid sample sandwiched between the            first plate and the second plate is heated at a rate of at            least 30° C./sec, and        -   wherein at least the portion of the liquid sample sandwiched            between the first plate and the second plate is cooled at a            rate of at least 30° C./sec when the heating/cooling layer            is not receiving the electromagnetic radiation generated by            the optical source, and        -   wherein the system consumes less than 500 mW of power.    -   2. The system of any prior embodiment, wherein the temperature        sensor is an image-based temperature sensor.    -   3. The system of any prior embodiment, wherein the temperature        sensor's field of view is 1 mm², 10 mm², 100 mm², 1000 mm², or        in a range between any of the two values.    -   4. The system of any prior embodiment, wherein the temperature        sensor's resolution is 1 um, 10 um, 100 um, 1 mm, or in a range        between any of the two values.    -   5. The system of any prior embodiment, wherein the working        thermal radiation wavelength of the temperature sensor's falls        in the range of 1 um to 10 um or 10 um to 100 um.    -   6. The system of any prior embodiment, wherein the thermal        sensor has a least a lens.    -   7. The system of any prior embodiment, wherein the thermal        sensor is an imager that can image at least a part of the        sample.    -   8. A method for measuring temperature of sample liquid in assay        device, comprising:        -   imaging the heating area in the assay device under thermal            imager;        -   segmenting air bubble area or defect area and sample liquid            area in the image;        -   measuring the temperature of sample liquid area.            E. Real Time Detection (qPCR) Setup

In certain embodiments, a system comprise a assay device, the heater,and an optical monitor to monitor an optical signal from a sample in thecard, wherein the optical signal give an indication of a nucleic acidamplification inside the Q-card and the optical signal is monitoredduring a PCR process (that is a real time PCR).

In certain embodiments, the optical monitor is a photodetector. Incertain embodiments, the optical monitor is one or more imagers thatimage an area or a volume of the sample. Hence the image gives anoptical signal in each location of the sample being imaged. An analysisof the optical signal image can give more accurate analysis on thenucleic acid amplification than a lump-sum optical signal detection.

In certain embodiments, the nucleic amplification during a PCR processis monitored by an imager or more imagers, where the imagers image anarea or a volume of a sample and the signal of the imager represents thenucleic acid amplification by the PCR. In certain embodiments, signal isfluorescence signal. In certain embodiments, signal is a color signal.

The terms “assay device” and “sample holder” are interchangeable.

In the optical signal image analysis, in certain embodiments, artificialintelligence is used. In the optical signal image analysis, in certainembodiments, machine learning is used.

In certain embodiments, the system does real-time PCR by adding one ormulti fluorescent excitation light sources and detectors into theheating and temperature monitoring system.

In certain embodiments, a system has imagers for sample temperatureimaging and for nucleic acid amplification signals monitoring imaging.In certain embodiments, the sample temperature imaging and the nucleicacid amplification signal monitoring imaging uses a single opticalmonitor.

Some embodiments have a real-time PCR system comprising a heating sourceand temperature monitoring system as described FIG. 11 and a pair offluorescent excitation light source and detector. In this case, theexcitation light source and fluorescence detector are on top of theassay device and aligned to the same excitation and detection area onthe assay device.

Some enbedments have a real-time PCR system comprising a heating source,a fan and temperature detector as a temperature control system and apair of fluorescent excitation light source (with filter) and detector(with lens and filter) as the real time detection system; bothtemperature control system and real time detection system are controlledby Programmable logic controller (PLC). The PLC is further controlled byan interface installed on a smartphone.

-   -   1. A system, comprising:        -   a device, comprising:        -   a first plate comprising a polymer material and having a            thickness less than or equal to 100 μm,        -   a second plate comprising a polymer material and having a            thickness less than or equal to 100 μm, wherein the second            plate is separated from the first plate in a parallel            arrangement by a distance less than or equal to the            thickness of the second plate,        -   a heating/cooling layer disposed on either the first plate            or the second plate, the heating/cooling layer having a            thickness and a thermal conductivity between 6×10⁻⁵ W/K            multiplied by the thickness of the heating/cooling layer and            1.5×10⁻⁴ W/K multiplied by the thickness of the            heating/cooling layer, and        -   a support frame configured to support at least one of the            first plate and the second plate;        -   an optical source configured to direct electromagnetic            radiation towards the heating/cooling layer,        -   a temperature sensor to monitor the temperature of the            heating area in the device;        -   a fluorescent excitation light source;        -   a fluorescent detector;        -   wherein the heating/cooling layer is configured to absorb at            least a portion of the electromagnetic radiation such that            at least a portion of a liquid sample sandwiched between the            first plate and the second plate is heated at a rate of at            least 30° C./sec, and        -   wherein at least the portion of the liquid sample sandwiched            between the first plate and the second plate is cooled at a            rate of at least 30° C./sec when the heating/cooling layer            is not receiving the electromagnetic radiation generated by            the optical source.

The system of any prior embodiment, wherein the excitation light sourcecan be but not limited to be a laser. The system of any priorembodiment, wherein the excitation light source can be but not limitedto be a LED. The system of any prior embodiment, wherein the fluorescentdetector is a photodetector. The system of any prior embodiment, whereinthe fluorescent detector is mounted on an optical tube. The system ofany prior embodiment, wherein the fluorescent detector is an image-basedsensor.

A method for measuring fluorescence signal of sample liquid in assaydevice, comprising:

-   -   imaging the heating area in the assay device under thermal        imager;    -   segmenting air bubble area or defect area and sample liquid area        in the image;    -   measuring the signal of sample liquid area.        A method for measuring fluorescence signal of sample liquid in        assay device,    -   the time of measuring fluorescence signal is at the primer        annealing and extension of each cycle.        A method for measuring fluorescence signal of sample liquid in        assay device, the time of measuring fluorescence signal is at        the end of primer annealing and extension of each cycle.        A method for measuring fluorescence signal of sample liquid in        assay device, the time of measuring fluorescence signal is at        the time of heating source is off in each cycle.

In certain embodiments, an imager (either for temperature sensing or fornucleic acid amplification monitoring) in the present invention, isconnected to a computer, where various signal processing techniques,including machine learning is used. In certain embodiments, the signalprocessing results will be used to control the heating to the sample.

F. Heating Optical Pipe Structure

In certain embodiments, an optical pipe (also termed opticalcollimator), that collimates the light of a light source into theheating zone/plate, comprises a hollow tube with a reflective wall.

One embodiment of an optical pipe comprises a hollow structure (e.g.,hollow tube) of round circle, rectangle, hexagonal, polygon, elliptic orcombination thereof.

One preferred embodiment of an optical pipe comprises a hexagonal hollowstructure.

One embodiment of an optical pipe comprises a hollow tube with areflective wall (i.e., its inner wall, outer wall, or both reflective).The reflective wall can be a thin light reflective coating on a wall ofthe hollow tube. The reflective coating can be a thin metal film, suchas gold, aluminum, silver, copper, or any mixture or combinationthereof.

In certain embodiments, the hollow structure is made of a dielectricmaterial of glasses, plastics, or a combination. In certain embodiments,the hollow structure is made of a metallic material.

Some embodiments have a round heating tube and a hexagonal heating tubewith a diameter of 6 mm and a point LED light source at the center ofone tube end. (b) shows the optical beam intensity measured at the otherend of tube. Clearly the hexagonal heating tube provides a more uniformdistribution of heating light intensity in the central 6 mm area.

In some embodiments, the hollow pipe has a length in the range of 1 mmto 70 mm, an inner dimension (diameter or width) in the range of 1 mm to40 mm, and a wall thickness in the range of 0.01 mm to 10 mm.

In some preferred embodiments, the hollow pipe for the light pipe has aninner diameter (or an average width) in a range of 1 mm to 5 mm, 5 mm to10 mm, 10 mm to 15 mm, 15 mm to 20 mm, 20 mm to 30 mm, or 30 mm to 50mm.

In some preferred embodiments, the hollow pipe for the light pipe has awall thickness (or an average width) in a range of 0.001 mm to 0.01 mm,0.01 mm to 0.1 mm, 0.1 mm to 0.5 mm, 0.5 mm to 1 mm, 1 mm to 2 mm, or 2mm to 50 mm.

Example. Fast SNAP PCR Amplification of PUC57 Plasmid DNA

The present technology uses the disclosed system for the PCRamplification of PUC57 plasmid DNA. The PCR reaction mixture wasprepared by mixing 10 uM PUC57 Forward primer, 10 uM PUC57 Reverseprimer and Cy5 labeled DNA probe with DNA buffer, 2.5 U/uL AptataqPolymerase, 25 mM MgCl2, dNTP, additives as Betaine, bovine serumalbumin (BSA), template DNA and ddH2O. 5 uL to 10 uL of the reaction wasadded onto the SNAP card and sealed for amplification.

In certain case, the whole card is incubated with 1% NaOH for 2 hoursunder 37° C., then washed with deionized water, then incubated with 4%bovine serum albumin (BSA) overnight under 4° C., washed with deionizedwater and dried at room temperature.

After amplification, the card is open and the production liquid issucked out for Gel electrophoresis analyze.

Our experiments have achieved a working SNAP PCR amplification ofnucleic acid (E-coli plasmid DNA) with assay device demonstrating (a)4.5 sec thermal cycling time (1 sec heating time from 60° C. to 95° C.,0.5 sec staying at 95° C., 2.5 sec cooling time from 95° C. to 60° C.,and 0.5 sec staying at 60° C.); (b) Gel electrophoresis results of SNAPPCR products ran in 3 minutes (40 cycles) and conventional PCR products(40 cycles) ran in 40 minutes shows 3 min SNAP PCR has a comparableamplification performance as 40 min conventional PCR. The M line in thefigure is a Gel electrophoresis marker with 100 bp line marked. BothSNAP PCR and conventional PCR have clear 100 bp production line andsimilar intensity. Negative sample without template does not show bar ingel analyze.

Applications

The devices, systems, and methods herein disclosed can be used invarious types of biological/chemical sampling, sensing, assays andapplications, which include the applications listed, described andsummarized in PCT Application (designating U.S.) No. PCT/US2016/046437,which was filed on Aug. 10, 2016, and is hereby incorporated byreference by its entirety.

In some embodiments, the devices, systems, and methods herein disclosedare used in a variety of different application in various field, whereindetermination of the presence or absence, quantification, and/oramplification of one or more analytes in a sample are desired. Forexample, in certain embodiments the subject devices, systems, andmethods are used in the detection of proteins, peptides, nucleic acids,synthetic compounds, inorganic compounds, and other molecules,compounds, mixtures and substances. The various fields in which thesubject devices, systems, and methods can be used include, but are notlimited to: diagnostics, management, and/or prevention of human diseasesand conditions, diagnostics, management, and/or prevention of veterinarydiseases and conditions, diagnostics, management, and/or prevention ofplant diseases and conditions, agricultural uses, food testing,environments testing and decontamination, drug testing and prevention,and others.

The applications of the present invention include, but are not limitedto: (a) the detection, purification, quantification, and/oramplification of chemical compounds or biomolecules that correlates withcertain diseases, or certain stages of the diseases, e.g., infectiousand parasitic disease, injuries, cardiovascular disease, cancer, mentaldisorders, neuropsychiatric disorders and organic diseases, e.g.,pulmonary diseases, renal diseases, (b) the detection, purification,quantification, and/or amplification of cells and/or microorganism,e.g., virus, fungus and bacteria from the environment, e.g., water,soil, or biological samples, e.g., tissues, bodily fluids, (c) thedetection, quantification of chemical compounds or biological samplesthat pose hazard to food safety, human health, or national security,e.g. toxic waste, anthrax, (d) the detection and quantification of vitalparameters in medical or physiological monitor, e.g., glucose, bloodoxygen level, total blood count, (e) the detection and quantification ofspecific DNA or RNA from biological samples, e.g., cells, viruses,bodily fluids, (f) the sequencing and comparing of genetic sequences inDNA in the chromosomes and mitochondria for genome analysis or (g) thedetection and quantification of reaction products, e.g., duringsynthesis or purification of pharmaceuticals.

In some embodiments, the subject devices, systems, and methods are usedin the detection of nucleic acids, proteins, or other molecules orcompounds in a sample. In certain embodiments, the devices, systems, andmethods are used in the rapid, clinical detection and/or quantificationof one or more, two or more, or three or more disease biomarkers in abiological sample, e.g., as being employed in the diagnosis, prevention,and/or management of a disease condition in a subject. In certainembodiments, the devices, systems, and methods are used in the detectionand/or quantification of one or more, two or more, or three or moreenvironmental markers in an environmental sample, e.g. sample obtainedfrom a river, ocean, lake, rain, snow, sewage, sewage processing runoff,agricultural runoff, industrial runoff, tap water or drinking water. Incertain embodiments, the devices, systems, and methods are used in thedetection and/or quantification of one or more, two or more, or three ormore foodstuff marks from a food sample obtained from tap water,drinking water, prepared food, processed food or raw food.

In some embodiments, the devices, systems and methods of the inventioncan be used to detect an analyte. In some embodiments, the analyte is apathogen. Exemplary pathogens that can be detected include, but are notlimited to: Varicella zoster; Staphylococcus epidermidis, Escherichiacoli, methicillin-resistant Staphylococcus aureus (MSRA), Staphylococcusaureus, Staphylococcus hominis, Enterococcus faecalis, Pseudomonasaeruginosa, Staphylococcus capitis, Staphylococcus warneri, Klebsiellapneumoniae, Haemophilus influenzae, Staphylococcus simulans,Streptococcus pneumoniae and Candida albicans; gonorrhea (Neisseriagorrhoeae), syphilis (Treponena pallidum), chlamydia (Chlamydiatracomitis), nongonococcal urethritis (Ureaplasm urealyticum), chancroid(Haemophilus ducreyi), trichomoniasis (Trichomonas vaginalis);Pseudomonas aeruginosa, methicillin-resistant Staphlococccus aureus(MSRA), Klebsiella pneumoniae, Haemophilis influenzae, Staphylococcusaureus, Stenotrophomonas maltophilia, Haemophilis parainfluenzae,Escherichia coli, Enterococcus faecalis, Serratia marcescens,Haemophilis parahaemolyticus, Enterococcus cloacae, Candida albicans,Moraxiella catarrhalis, Streptococcus pneumoniae, Citrobacter freundii,Enterococcus faecium, Klebsella oxytoca, Pseudomonas fluorscens,Neiseria meningitidis, Streptococcus pyogenes, Pneumocystis carinii,Klebsella pneumoniae, Legionella pneumophila, Mycoplasma pneumoniae, andMycobacterium tuberculosis, etc.

In some embodiments, the devices, systems and methods of the inventioncan be used to detect an analyte that is a diagnostic marker.

In some embodiments, the invention is directed to a kit containing adevice of the invention. In some embodiments, the kit includes a deviceconfigured to specifically bind an analyte described herein. In someembodiments, the kit includes instructions for practicing the subjectmethods using a hand held device, e.g., a mobile phone. In someembodiments, the instructions can be present in the kits in a variety offorms, one or more of which can be present in the kit. One form in whichthese instructions can be present is as printed information on asuitable medium or substrate, e.g., a piece or pieces of paper on whichthe information is printed, in the packaging of the kit, in a packageinsert, etc. Another means would be a computer readable medium, e.g.,diskette, CD, DVD, Blu-Ray, computer-readable memory, etc., on which theinformation has been recorded or stored. Yet another means that can bepresent is a website address which can be used via the Internet toaccess the information at a removed site. The kit can further include asoftware for implementing a method for measuring an analyte on a device,as described herein, provided on a computer readable medium. Anyconvenient means can be present in the kits.

In some embodiments, the kit includes a detection agent that includes adetectable label, e.g. a fluorescently labeled antibody oroligonucleotide that binds specifically to an analyte of interest, foruse in labeling the analyte of interest. The detection agent can beprovided in a separate container as the device, or can be provided inthe device.

In some embodiments, the kit includes a control sample that includes aknown detectable amount of an analyte that is to be detected in thesample. The control sample can be provided in a container, and can be insolution at a known concentration, or can be provided in dry form, e.g.,lyophilized or freeze dried. The kit can also include buffers for use indissolving the control sample, if it is provided in dry form.

In some embodiments, the devices, systems and methods of the inventioncan be used for simple, rapid blood cell counting using a smartphone. Insome embodiments, the first plate and the second plate are selected froma thin glass slide (e.g., 0.2 mm thick) or a thin plastic film (e.g., 15mm thick) of a relative flat surface, and each have an areas with alength and width in about 0.5 cm to 10 cm. In some embodiments, thespacers are made of glass, plastics, or other materials that would notdeform significantly under a pressing. In some embodiments, before thesample deposition, the spacers are placed on the first plate, the secondplate or both; and the first plate, the second plate or both areoptionally coated with reagent that facilitate the blood counting(staining dyes and/or anticoagulant). In some embodiments, the firstplate and the second plate can be sealed in a bag for easy transport andlonger shelf life-time.

In some embodiments of blood cell count testing, only about 1 uL(microliter) (or about 0.1 uL to 3 uL) of blood is needed for thesample, which can be taken, for example, from a finger or other humanbody location. In some embodiments, the blood sample can be directlydeposited from human body (e.g., finger) onto the first plate and thesecond plate, without any dilution. In such embodiments, the first plateand the second plate can face each other, so that blood sample isbetween the inner surfaces of the first plate and the second plate. Insome embodiments, reagents are pre-deposited (staining dyes oranticoagulant), they are deposited on the inner surface for mixing withthe sample. The first plate and the second plate can then be pressed byfingers or a simple mechanical device (e.g. a clip that presses using aspring). Under the press, the inner spacing is reduced, the reductionwill be eventually stopped at the value set by the spacers' height andthe final sample thickness is reached, which generally is equal to thefinal inner spacing. Since the final inner spacing is known, the finalsample thickness become known, namely being quantified (measured) bythis method.

In some embodiments, if the blood sample is not diluted, after pressing(sample deformation) the spacers and hence the final sample thicknesscan be thin, e.g., less than 1 um, less than 2 um, less than 3 um, lessthan 4 um, less than 5 um, less than 7 um, less than 10 um, less than 15um, less than 20 um, less than 30 um, less than 40 um, less than 50 um,less than 60 um, less than 80 um, less than 100 um, less than 150 um, orany ranges between any of the two numbers. A thin final sample can beuseful because if the final sample thickness is thick, then many redcells can overlap during the imaging, which can make the cell countinginaccurate. For example, about 4 um thick of whole blood withoutdilution will give about one layer of blood red cells.

After the pressing, the sample can be imaged by a smartphone eitherdirectly or through an additional optical elements (e.g. lenses,filters, or light sources as needed). The image of the sample can beprocessed to identify the types of the cells as well as the cell number.The image processing can be done locally at the same smartphone thattakes the image or remotely but the final result transmitted back to thesmartphone (where the image is transmitted to a remote location and isprocessed there.) The smart phone will display the cell number for aparticular cell. In some cases, certain advices will be displayed. Theadvices can stored on the smartphone before the test or come from aremote machines or professionals.

In certain embodiments, reagents are placed on the inner surfaces of thefirst plate and/or the second plate using the methods and devicesdescribed herein.

In some embodiments, a device or a method for the blood testingcomprises (a) a device or a method described herein and (b) a platespacing (i.e. the distance between the inner surfaces of the two plates)at the closed configuration or a use of such spacing, wherein aundiluted whole blood in the plate-spacing has an average inter-celldistance in the lateral direction for the red blood cells (RBC) largerthan the average diameter of the disk shape of the RBC.

In some embodiments, a device or a method to arrange the orientation ofa non-spherical cell comprises (a) a device or a method in as describedherein and (b) a plate spacing (i.e. the distance between the innersurfaces of the two plates) at the closed configuration or a use of suchspacing, wherein the spacing less than the average size of the cell inits long direction (the long direction is the maximum dimensiondirection of a cell). Such arrangement can improve the measurements ofthe sample volume (e.g. red blood cell volume).

In some embodiments, the analytes in the blood tests include proteinmarkers, a list of which can be found at the website of the AmericanAssociation for Clinical Chemistry).

In some embodiments, the devices, systems and methods of the inventioncan be used to detect or diagnose a health condition. In someembodiments, the health condition includes, but is not limited to:chemical balance; nutritional health; exercise; fatigue; sleep; stress;prediabetes; allergies; aging; exposure to environmental toxins,pesticides, herbicides, synthetic hormone analogs; pregnancy; menopause;and andropause.

In some embodiments, relative levels of nucleic acids in two or moredifferent nucleic acid samples can be obtained using such methods, andcompared. In these embodiments, the results obtained from the methodsdescribed herein are usually normalized to the total amount of nucleicacids in the sample (e.g., constitutive RNAs), and compared. This can bedone by comparing ratios, or by any other means. In particularembodiments, the nucleic acid profiles of two or more different samplescan be compared to identify nucleic acids that are associated with aparticular disease or condition.

In some embodiments, the devices, systems and methods in the presentinvention can include a) obtaining a sample, b) applying the sample todevice containing a capture agent that binds to an analyte of interest,under conditions suitable for binding of the analyte in a sample to thecapture agent, c) washing the device, and d) reading the device, therebyobtaining a measurement of the amount of the analyte in the sample. Insome embodiments, the analyte can be a biomarker, an environmentalmarker, or a foodstuff marker. The sample in some instances is a liquidsample, and can be a diagnostic sample (such as saliva, serum, blood,sputum, urine, sweat, lacrima, semen, or mucus); an environmental sampleobtained from a river, ocean, lake, rain, snow, sewage, sewageprocessing runoff, agricultural runoff, industrial runoff, tap water ordrinking water; or a foodstuff sample obtained from tap water, drinkingwater, prepared food, processed food or raw food. In some embodiments,the device can be placed in a microfluidic device and the applying stepb) can include applying a sample to a microfluidic device comprising thedevice. In some embodiments, the reading step d) can include detecting afluorescence or luminescence signal from the device. In someembodiments, the reading step d) can include reading the device with ahandheld device configured to read the device. The handheld device canbe a mobile phone, e.g., a smart phone. In some embodiments, the devicecan include a labeling agent that can bind to an analyte-capture agentcomplex on the device. In some embodiments, the devices, systems andmethods in the present invention can further include, between steps c)and d), the steps of applying to the device a labeling agent that bindsto an analyte-capture agent complex on the device, and washing thedevice. In any embodiment, the reading step d) can include reading anidentifier for the device. The identifier can be an optical barcode, aradio frequency ID tag, or combinations thereof. In some embodiments,the devices, systems and methods in the present invention can furtherinclude applying a control sample to a control device containing acapture agent that binds to the analyte, wherein the control sampleincludes a known detectable amount of the analyte, and reading thecontrol device, thereby obtaining a control measurement for the knowndetectable amount of the analyte in a sample. In some embodiments, thesample can be a diagnostic sample obtained from a subject, the analytecan be a biomarker, and the measured amount of the analyte in the samplecan be diagnostic of a disease or a condition.

In some embodiments, the devices, systems and methods in the presentinvention can further include receiving or providing to the subject areport that indicates the measured amount of the biomarker and a rangeof measured values for the biomarker in an individual free of or at lowrisk of having the disease or condition, wherein the measured amount ofthe biomarker relative to the range of measured values is diagnostic ofa disease or condition. In some embodiments, the devices, systems andmethods in the present invention can further include diagnosing thesubject based on information including the measured amount of thebiomarker in the sample. In some embodiments, the diagnosing stepincludes sending data containing the measured amount of the biomarker toa remote location and receiving a diagnosis based on informationincluding the measurement from the remote location. In some embodiments,the biomarker can be selected from those listed in the Tables. In someembodiments, the device can contain a plurality of capture agents thateach binds to a biomarker described herein, wherein the reading step d)includes obtaining a measure of the amount of the plurality ofbiomarkers in the sample, and wherein the amount of the plurality ofbiomarkers in the sample is diagnostic of a disease or condition. Insome embodiments, the capture agent can be an antibody epitope and thebiomarker can be an antibody that binds to the antibody epitope. In someembodiments, the antibody epitope includes a biomolecule, or a fragmentthereof, selected from the Tables. In some embodiments, the antibodyepitope includes an allergen, or a fragment thereof, selected from theTables. In some embodiments, the antibody epitope includes an infectiousagent-derived biomolecule, or a fragment thereof, selected from Tables.In some embodiments, the device can contain a plurality of antibodyepitopes selected from the Tables, wherein the reading step d) includesobtaining a measure of the amount of a plurality of epitope-bindingantibodies in the sample, and wherein the amount of the plurality ofepitope-binding antibodies in the sample is diagnostic of a disease orcondition.

In some embodiments, the sample can be an environmental sample, andwherein the analyte can be an environmental marker. In some embodiments,the environmental marker described herein. In some embodiments, themethod can include receiving or providing a report that indicates thesafety or harmfulness for a subject to be exposed to the environmentfrom which the sample was obtained. In some embodiments, the method caninclude sending data containing the measured amount of the environmentalmarker to a remote location and receiving a report that indicates thesafety or harmfulness for a subject to be exposed to the environmentfrom which the sample was obtained. In any embodiment, the device caninclude a plurality of capture agents that each binds to anenvironmental marker described herein, and wherein the reading step d)can include obtaining a measure of the amount of the plurality ofenvironmental markers in the sample.

In some embodiments, the sample can be a foodstuff sample, wherein theanalyte can be a foodstuff marker, and wherein the amount of thefoodstuff marker in the sample can correlate with safety of thefoodstuff for consumption. In some embodiments, the foodstuff marker isan example described herein. In any embodiment, the method can includereceiving or providing a report that indicates the safety or harmfulnessfor a subject to consume the foodstuff from which the sample isobtained. In any embodiment, the method can include sending datacontaining the measured amount of the foodstuff marker to a remotelocation and receiving a report that indicates the safety or harmfulnessfor a subject to consume the foodstuff from which the sample isobtained. In any embodiment, the device array can include a plurality ofcapture agents that each binds to a foodstuff marker described herein,wherein the obtaining can include obtaining a measure of the amount ofthe plurality of foodstuff markers in the sample, and wherein the amountof the plurality of foodstuff marker in the sample can correlate withsafety of the foodstuff for consumption.

In some embodiments, the subject device is part of a microfluidicdevice. In some embodiments, the subject devices, systems, and methodsare used to detect a fluorescence or luminescence signal. In someembodiments, the subject devices, systems, and methods include, or areused together with, a communication device, such as but not limited to:mobile phones, tablet computers and laptop computers. In someembodiments, the subject devices, systems, and methods include, or areused together with, an identifier, such as but not limited to an opticalbarcode, a radio frequency ID tag, or combinations thereof.

In some embodiments, the sample is a diagnostic sample obtained from asubject, the analyte is a biomarker, and the measured amount of theanalyte in the sample is diagnostic of a disease or a condition. In someembodiments, the subject devices, systems and methods further includereceiving or providing to the subject a report that indicates themeasured amount of the biomarker and a range of measured values for thebiomarker in an individual free of or at low risk of having the diseaseor condition, wherein the measured amount of the biomarker relative tothe range of measured values is diagnostic of a disease or condition.

In some embodiments, the sample is an environmental sample, and whereinthe analyte is an environmental marker. In some embodiments, the subjectdevices, systems and methods includes receiving or providing a reportthat indicates the safety or harmfulness for a subject to be exposed tothe environment from which the sample was obtained. In some embodiments,the subject devices, systems and methods include sending data containingthe measured amount of the environmental marker to a remote location andreceiving a report that indicates the safety or harmfulness for asubject to be exposed to the environment from which the sample wasobtained.

In some embodiments, the sample is a foodstuff sample, wherein theanalyte is a foodstuff marker, and wherein the amount of the foodstuffmarker in the sample correlate with safety of the foodstuff forconsumption. In some embodiments, the subject devices, systems andmethods include receiving or providing a report that indicates thesafety or harmfulness for a subject to consume the foodstuff from whichthe sample is obtained. In some embodiments, the subject devices,systems and methods include sending data containing the measured amountof the foodstuff marker to a remote location and receiving a report thatindicates the safety or harmfulness for a subject to consume thefoodstuff from which the sample is obtained.

Various samples can be used in the assays conducted with the devices,apparatus, and systems herein described. In some embodiments, the samplecomprises nucleic acids. In some embodiments, the sample comprisesproteins. In some embodiments, the sample carbohydrates. The currentdevices, apparatus, and systems can be used to rapidly change thetemperature of the sample and steadily maintain the temperature of thesample, providing a fast and cost-effective approach to process samples.In addition, various applications (e.g. assays) can be conducted withthe devices, apparatus, and systems herein described. Such applicationsinclude but are not limited to diagnostic testing, health monitoring,environmental testing, and/or forensic testing. Such applications alsoinclude but are not limited to various biological, chemical, andbiochemical assays (e.g. DNA amplification, DNA quantification,selective DNA isolation, genetic analysis, tissue typing, oncogeneidentification, infectious disease testing, genetic fingerprinting,and/or paternity testing).

In some embodiments, the “sample” can be any nucleic acid containing ornot containing samples, including but not limited to human bodilyfluids, such as whole blood, plasma, serum, urine, saliva, and sweat,and cell cultures (mammalian, plant, bacteria, fungi). The sample can befreshly obtained, or stored or treated in any desired or convenient way,for example by dilution or adding buffers, or other solutions orsolvents. Cellular structures can exist in the sample, such as humancells, animal cells, plant cells, bacteria cells, fungus cells, andvirus particles.

The term “nucleic acid” as used herein refers to any DNA or RNAmolecule, or a DNA/RNA hybrid, or mixtures of DNA and/or RNA. The term“nucleic acid” therefore is intended to include but not limited togenomic or chromosomal DNA, plasmid DNA, amplified DNA, cDNA, total RNA,mRNA and small RNA. The term “nucleic acid” is also intended to includenatural DNA and/or RNA molecule, or synthetic DNA and/or RNA molecule.In some embodiments, cell-free nucleic acids are presence in the sample,as used herein “cell-free” indicates nucleic acids are not contained inany cellular structures. In some other embodiments, nucleic acids arecontained within cellular structures, which include but not limited tohuman cells, animal cells, plant cells, bacterial cells, fungi cells,and/or viral particles. Nucleic acids either in the form of cell-freenucleic acids or within cellular structures or a combination thereof,can be presence in the sample. In some further embodiments, nucleicacids are purified before introduced onto the inner surface of the firstplate. In yet further embodiments, nucleic acids can be within a complexassociated with other molecules, such as proteins and lipids.

The method of the invention is suitable for samples of a range ofvolumes. Sample having different volumes can be introduced onto theplates having different dimensions.

As used herein, “nucleic acid amplification” includes any techniquesused to detect nucleic acids by amplifying (generating numerous copiesof) the target molecules in samples, herein “target” refers to asequence, or partial sequence, of nucleic acid of interest. Suitablenucleic acid amplification techniques include but not limited to,different polymerase chain reaction (PCR) methods, such as hot-startPCR, nested PCR, touchdown PCR, reverse transcription PCR, RACE PCR,digital PCR, etc., and isothermal amplification methods, such asLoop-mediated isothermal amplification (LAMP), strand displacementamplification, helicase-dependent amplification, nicking enzymeamplification, rolling circle amplification, recombinase polymeraseamplification, etc.

As used herein, “necessary reagents” or “reagents” include but are notlimited to, primers, deoxynucleotides (dNTPs), bivalent cations (e.g.Mg2+), monovalent cation (e.g. K+), buffer solutions, enzymes,additives, and reporters. “Necessary reagents for nucleic acidamplification” or “reagents for nucleic acid amplification” can beeither in the dry form on the inner surface of the first or the secondplate or both, or in a liquid form encased in, embedded in, orsurrounded by, a material that melts with increasing temperatures, suchas, for example, paraffin.

As used herein, “primers”, in some embodiments, can refer to a pair offorward and reverse primers. In some embodiments, primers can refer to aplurality of primers or primer sets. As used herein, enzymes suitablefor nucleic acid amplification include, but not limited to,DNA-dependent polymerase, or RNA-dependent DNA polymerase, orDNA-dependent RNA polymerase. Examples of suitable DNA-dependentpolymerases include but not limited to AptaTaq polymerase, Kapa2G Fastpolymerase, Kapa2G Robust, Z-Taq polyermase, Terra PCR DirectPolymerase, SpeedStar HS DNA polymerase, Phusion DNA polymerase, andHigh-Fidelity DNA polymerase.

As used herein, “additives”, in some embodiments, include but notlimited to, 7-deaza-2′-deoxyguanosine 7-deaza dGTP, BSA, gelatin,betaine, DMSO, formamide, Tween 20, NP-40, Triton X-100,tetramethylammonium chloride.

As used herein, the term “reporter” refers to any tag, label, or dyethat can bind to, or intercalate within, the nucleic acid molecule or beactivated by byproducts of the amplification process to enablevisualization of the nucleic acid molecule or the amplification process.Suitable reporters include but are not limited to fluorescent labels ortags or dyes, intercalating agents, molecular beacon labels, orbioluminescent molecules, or a combination thereof.

In some other embodiments, as used herein, “necessary reagents” or“reagents” (e.g., for nucleic acid amplification reactions) can alsoinclude cell lysing reagent, which facilitates to break down cellularstructures. Cell lysing reagents include but not limited to salts,detergents, enzymes, and other additives. The term “salts” hereininclude but not limited to lithium salt (e.g. lithium chloride), sodiumsalt (e.g. sodium chloride), potassium (e.g. potassium chloride). Theterm “detergents” herein can be ionic, including anionic and cationic,non-ionic or zwitterionic. The term “ionic detergent” as used hereinincludes any detergent which is partly or wholly in ionic form whendissolved in water. Suitable anionic detergents include but not limitedto sodium dodecyl sulphate (SDS) or other alkali metal alkylsulphatesalts or similar detergents, sarkosyl, or combinations thereof. The term“enzymes” herein include but not limited to lysozyme, cellulase, andproteinase. In addition, chelating agents including but not limited toEDTA, EGTA and other polyamino carboxylic acids, and some reducingagents, such as dithiotreitol (dTT), can also be included in cell lysingreagents. The compositions of necessary reagents herein vary accordingto rational designs of different amplification reactions. In someembodiments, for example when conducting isothermal amplification viaLAMP, the sample is heated to 60-65° C. for about 1-70 min.

As used herein, “nucleic acid amplification product” refers to variousnucleic acids generated by nucleic acid amplification techniques. Typesof nucleic acid amplification products herein include but not limited tosingle strand DNA, single strand RNA, double strand DNA, linear DNA, orcircular DNA, etc. In some embodiments, nucleic acid amplificationproduct can be identical nucleic acids having the same length andconfiguration. In some other embodiments, nucleic acid amplificationproducts can be a plurality of nucleic acids having different lengthsand configurations.

In some embodiments, nucleic acids accumulated after nucleic acidamplification is quantified using reporters. As defined and used above,reporter having quantifiable features that is correlated with thepresence or the absence, or the amount of the nucleic acid ampliconsaccumulated in the closed chamber.

As used herein, “cell lysing reagents”, intend to include but notlimited to salts, detergents, enzymes, and other additives, whichfacilitates to disrupt cellular structures. The term “salts” hereininclude but not limited to lithium salt (e.g. lithium chloride), sodiumsalt (e.g. sodium chloride), potassium (e.g. potassium chloride). Theterm “detergents” herein can be ionic, including anionic and cationic,non-ionic or zwitterionic. The term “ionic detergent” as used hereinincludes any detergent which is partly or wholly in ionic form whendissolved in water. Suitable anionic detergents include but not limitedto sodium dodecyl sulphate (SDS) or other alkali metal alkylsulphatesalts or similar detergents, sarkosyl, or combinations thereof. The term“enzymes” herein include but not limited to lysozyme, cellulase, andproteinase. In addition, chelating agents including but not limited toEDTA, EGTA and other polyamino carboxylic acids, and some reducingagents, such as dithiotreitol (dTT), can also be included in cell lysingreagents. The compositions of necessary reagents herein vary accordingto rational designs of different amplification reactions.

As used herein, “necessary reagent 2” include but not limited to,primers, deoxynucleotides (dNTPs), bivalent cations (e.g. Mg2+),monovalent cation (e.g. K+), buffer solutions, enzymes, and reporters.Necessary reagent 2 for nucleic acid amplification can be either in thedry form on the inner surface of the first or the second plate or both,or in a liquid form encased in, embedded in, or surrounded by, amaterial that melts with increasing temperatures, such as, for example,paraffin.

A Rapid Heating and Cooling Apparatus where a Separate Heating ElementOutside QMAX-Card

In some embodiments, the apparatus further comprises a separate heatingelement that is outside of RHC card and is configured to heat the RHCcard when being placed near or in contact with the RHC card. Theseparate heating element is capable of attaching or detaching a RHCcard, and gain energy from a heating source, in a similar fashion as theheating/cooling layer. The separate heating element allow a RHC cardwithout a heating/cooling layer.

The terms “CROF Card (or card)”, “COF Card”, “QMAX-Card”, “Q-Card”,“CROF device”, “COF device”, “QMAX-device”, “CROF plates”, “COF plates”,and “QMAX-plates” are interchangeable and may be used to identifyembodiments of the devices described herein. The term “X-plate” refersto one of the two plates in a CROF card, wherein the spacers are fixedto this plate. More descriptions of the COF Card, CROF Card, and X-plateare described in the provisional application Ser. No. 62/456,065, filedon Feb. 7, 2017, which is incorporated herein in its entirety for allpurposes.

A RHC card is a QMAX-care with or without spacer plus a heating/coolinglayer on or inside of one of the plate.

FIGS. 4A and 4B show perspective and sectional views of an embodiment ofthe device of the present invention. FIG. 9A illustrates the device(also termed “sample holder” of the system) 100 in an openconfiguration. As shown in FIG. 4A, the sample holder 100 comprises afirst plate 10, a second plate 20, and a spacing mechanism (not shown).The first plate 10 and second plate 20 respectively comprise an innersurface (11 and 21, respectively) and an outer surface (12 and 22,respectively). Each inner surface has a sample contact area (notindicated) for contacting a fluidic sample to be processed and/oranalyzed by the device.

The first plate 10 and the second plate 20 are movable relative to eachother into different configurations. One of the configurations is theopen configuration, in which, as shown in FIG. 9A, the first plate 10and the second plate 20 are partially or entirely separated apart, andthe spacing between the first plate 10 and the second plate 20 (i.e. thedistance between the first plate inner surface 11 and the second plateinner surface 21) is not regulated by the spacing mechanism. The openconfiguration allows a sample to be deposited on the first plate, thesecond plate, or both, in the sample contact area.

As shown in FIG. 9A, the second plate 20 further comprises aheating/cooling layer 112 in the sample contact area. It is alsopossible that the first plate 10 alternatively or additionally comprisethe heating/cooling layer 112. In some embodiments, the heating/coolinglayer 112 is configured to efficiently absorb radiation (e.g.electromagnetic waves) shed on it. The absorption percentage is 50% ormore, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more,99% or more, 100% or less, 85% or less, 75% or less, 65% or less, or 55%or less, or in a range between any of the two values. Theheating/cooling layer 112 is further configured to convert at least asubstantial portion of the absorbed radiation energy into heat (thermalenergy). For example, the heating/cooling layer 112 is configured toemit radiation in the form of heat after absorbing the energy fromelectromagnetic waves. The term “substantial portion” or “substantially”as used herein refers to a percentage that is 50% or more, 60% or more,70% or more, 80% or more, 90% or more, 95% or more, 99% or more, 99% ormore, or 99.9% or more.

FIGS. 8A and 3B illustrate the sample card in a closed configuration,where the heating/cooling layer comprises a heating zone that isdirectly being/to be heated by a heating source; FIG. 3A shows aprospective view and FIG. 3B shows a sectional view. In someembodiments, the heating/cooling layer comprises a heating zone that isbeing/to be directly heated by a heating source. In some embodiments,the heating sources emits electromagnetic radiation (waves) that, withor without modulation by lenses or other modulators, reaches theheating/cooling layer. The area that directly receives such radiation(waves) is referred to as the heating zone.

In some embodiments, the heating zone is smaller than the entire area ofthe heating/cooling layer. In some embodiments, the heating zone isabout 1/1000, 1/500, 1/200, 1/100, 1/50, 1/20, 1/10, 1/5, 1/2, or 2/3 ofthe area of the heating/cooling layer, or in a range between any of thetwo values. In some embodiments, when the sample is loaded andcompressed, by the two plates, into a thin layer, the volume of thesample directly in the path of the electromagnetic waves, or directly incontact with the area of the heating zone, is referred to as the heatedvolume. In some embodiments, since the sample layer is thin and/or dueto the superior absorption properties of the heating/cooling layer, thesample in the heated volume can be rapidly heated to a desiredtemperature. In some embodiments, the sample in the heated volume canalso be rapidly cooled to a desired temperature.

Biochemistry and Assays

The thermal cycler system and associated methods of the presentinvention can be used to facilitate a chemical, biological or medicalassay or reaction. In some embodiments, the reaction requirestemperature changes. In some embodiments, the reaction requires orprefers rapid temperature change in order to avoid non-specific reactionand/or reduce wait time. In certain embodiments, the system and methodsof the present invention is used to facilitate a reaction that requirescyclical temperature changes for amplification of a nucleotide in afluidic sample; such reactions include but are not limited to polymerasechain reaction (PCR). The descriptions below use PCR as an example toillustrate the capability and utilization of the thermal cycler systemand method of the present invention. It is should be noted, however,some embodiments of the device, systems and method herein described alsoapply to other assays and/or reactions that require temperature controland change.

In some embodiments, the assays (e.g. PCR) can be conducted with anon-processed sample. For example, the template of a PCR reaction can beprovided by a sample directed obtained from a subject without additionalprocessing. In some embodiments, the sample can be whole blood from anindividual. In some embodiments, such a “one-step” approach would allowfor more convenient use of the devices herein described.

In some embodiments, the sample 90 is a pre-mixed reaction medium forpolymerase chain reaction (PCR). For example, in certain embodiments,the reaction medium includes components such as but not limited to: DNAtemplate, two primers, DNA polymerase (e.g. Taq polymerase),deoxynucleoside triphosphates (dNTPs), bivalent cations (e.g. Mg²⁺),monovalent cation (e.g. K⁺), and buffer solution. The specificcomponents, the concentrations of each component, and the overall volumevaries according to rational design of the reaction. In someembodiments, the PCR assay requires a number of changes/alterations insample temperature between the following steps: (i) the optionalinitialization step, which requires heating the sample to 92-98° C.; (2)the denaturation step, which requires heating the sample to 92-98° C.;(3) the annealing step, which requires lowering the sample temperatureto 50-65° C.; (4) extension (or elongation) step, which requires heatingthe sample to 75-80° C.; (5) repeating steps (2)-(4) for about 20-40times; and (6) completion of the assay and lowering the temperature ofthe sample to ambient temperature (e.g. room temperature) or cooling toabout 4° C. The specific temperature and the specific time period foreach step varies and depends on a number of factors, including but notlimited to length of the target sequence, length of the primers, thecation concentrations, and/or the GC percentage.

The thermal cycler system of the present invention provides rapidtemperature change for the PCR assay. For example, referring to panels(A) and (B) of FIG. 3 and panel (B) of FIG. 9 , in some embodiments, thesample 90 (e.g. pre-mixed reaction medium) is added to one or both ofthe plates 10 and 20 in the open configuration and the plates isswitched to the closed configuration to compress the sample 90 into athin layer which has a thickness 102 that is regulated by a spacingmechanism (not shown); the heating source 202 projects anelectromagnetic wave 210 to the first plate 10 (e.g. specifically to theheating/cooling layer 112); the heating/cooling layer 112 is configuredto absorb the electromagnetic wave 210 and convert at least asubstantial portion of said electromagnetic wave 210 into heat, whichincreases the temperature of the sample; the removal of theelectromagnetic wave 210 results in a temperature decrease in the sample90.

In some embodiments, by projecting an electromagnetic wave 210 to theheating/cooling layer 112 or increasing the intensity of theelectromagnetic wave, the thermal cycler systems provide rapid heating(increase temperature) for any or all of the initialization step, thedenaturation step and/or the extension/elongation step; in someembodiments, with the removal of the electromagnetic wave projected fromthe heating source 202 or the decrease of the intensity of theelectromagnetic wave, the cooling to the annealing step and/or the finalcooling step is achieved with rapid speed. In some embodiments, theelectromagnetic wave 210 or an increase of the intensity of theelectromagnetic wave 210 creates an ascending temperature ramp rate ofat least 80° C./s, 70° C./s, 60° C./s, 50° C./s, 45° C./s, 40° C./s, 35°C./s, 30° C./s, 25° C./s, 20° C./s, 18° C./s, 16° C./s, 14° C./s, 12°C./s, 10° C./s, 9° C./s, 8° C./s, 7° C./s, 6° C./s, 5° C./s, 4° C./s, 3°C./s, or 2° C./s, or in a range between any of the two values. Incertain embodiments, the average ascending temperature ramp rate in aPCR assay is 10° C./s or more. In some embodiments, the removal of theelectromagnetic wave 210 or a reduction of the intensity of theelectromagnetic wave 210 results in a descending temperature ramp rateof at least 80° C./s, 70° C./s, 60° C./s, 50° C./s, 45° C./s, 40° C./s,35° C./s, 30° C./s, 25° C./s, 20° C./s, 18° C./s, 16° C./s, 1° C./s, 12°C./s, 10° C./s, 9° C./s, 8° C./s, 7° C./s, 6° C./s, 5° C./s, 4° C./s, 3°C./s, or 2° C./s, or in a range between any of the two values. Incertain embodiments, the average descending temperature ramp rate in aPCR assay is 5° C./s or more. As used here, the term “ramp rate” refersto the speed of temperature change between two pre-set temperatures. Insome embodiments, the average ascending or descending temperature toeach step is different.

During a PCR, within any step after the target temperature has beenreached, the sample needs to be maintained at the target temperature fora certain period of time. The thermal cycler system of the presentinvention provides the temperature maintenance function by (1) adjustingthe intensity of the electromagnetic wave 210, lowering it if thetemperature has been raised to the target or increasing it if thetemperature has been decreased to the target, and/or (2) keep the targettemperature by balancing the heat provided to the sample and the heatremoved from the sample.

Additional Exemplary Embodiments

AAA-1.1 A device for rapidly changing the temperature of a fluidicsample, comprising:

a first plate (10), a second plate (20), a heating layer (112-1), and acooling layer (112-2), wherein:

each of the first plate and the second plate has, on its respectiveinner surface, a sample contact area for contacting a fluidic sample;wherein the sample contact areas face each other, are separated by anaverage separation distance of 200 um or less between them, and arecapable of contacting the sample and sandwiching the sample betweenthem;

the heating layer is:

positioned on the inner surface, the outer surface, or inside of one ofthe plates, and

configured to heat a relevant volume of the sample, wherein the relevantvolume of the sample is a portion or an entirety of the sample that isbeing heated to a desired temperature; and

the cooling layer is:

positioned on the inner surface, the outer surface, or inside of one ofthe plates; and

configured to cool the relevant sample volume; and

comprises a layer of material that that has a thermal conductivity tothermal capacity ratio of 0.6 cm²/sec or larger;

wherein the distance between the cooling layer and a surface of therelevant sample volume is zero or less than a distance that isconfigured to make the thermal conductance per unit area between thecooling layer and the surface of the relevant sample volume equal to 70W/(m²·K) or larger; and

wherein, in some embodiments, the heating layer and cooling layer arethe same material layer that has a heating zone and a cooling zone, andwherein the heating zone and cooling zone can have the same area ordifferent areas.

AAA-1.2 A device for rapidly changing the temperature of a fluidicsample, comprising:

A first plate (10), a second plate (20), a heating layer (112-1), and acooling layer (112-2), wherein:

each of the first plate and the second plate has, on its respectiveinner surface, a sample contact area for contacting a fluidic sample;wherein the sample contact areas face each other, are separated by anaverage separation distance of 200 um or less from each other, and arecapable of contacting the sample and sandwiching the sample betweenthem; the heating layer is:

positioned on the inner surface, the outer surface, or inside of one ofthe plates, and configured to heat a relevant volume of the sample,wherein the relevant volume of the sample is a portion or an entirety ofthe sample that is being heated to a desired temperature; and

the cooling layer is:

positioned on the inner surface, the outer surface, or inside of one ofthe plates; and

configured to cool the relevant sample volume; and

comprises a layer of material that that has a thermal conductivity tothermal capacity ratio of 0.6 cm²/sec or larger, wherein the highthermal conductivity to thermal capacity ratio layer has an area largerthan the lateral area of the sample volume;

wherein the distance between the cooling layer and a surface of therelevant sample volume is zero or less than a distance that isconfigured to make the thermal conductance per unit area between thecooling layer and the surface of the relevant sample volume equal to 70W/(m²·K) or larger; and

wherein, in some embodiments, the heating layer and cooling layer arethe same material layer that has a heating zone and a cooling zone, andwherein the heating zone and cooling zone can have the same area ordifferent areas.

AAA-1.3 A device for rapidly changing the temperature of a fluidicsample, comprising:

a first plate (10), a second plate (20), a heating layer (112-1), and acooling layer (112-2), wherein:

the first and second plates are movable relative to each other intodifferent configurations;

each of the first plate and the second plate has, on its respectiveinner surface, a sample contact area for contacting a fluidic sample;wherein the sample contact areas face each other, are separated by anaverage separation distance of 200 um or less, and are capable ofsandwiching the sample between them;

the heating layer is:

positioned on the inner surface, the outer surface, or inside of one ofthe plates, and

configured to heat a relevant volume of the sample, wherein the relevantvolume of the sample is a portion or an entirety of the sample that isbeing heated to a desired temperature; and

the cooling layer is:

positioned on the inner surface, the outer surface, or inside of one ofthe plates; and

configured to cool the relevant sample volume; and

comprises a layer of material that that has a thermal conductivity tothermal capacity ratio of 0.6 cm²/sec or larger;

wherein the distance between the cooling layer and a surface of therelevant sample volume is zero or less than a distance that isconfigured to make the thermal conductance per unit area between thecooling layer and the surface of the relevant sample volume equal to 70W/(m²·K) or larger;

wherein one of the configurations is an open configuration, in which:the two plates are partially or completely separated apart and theaverage spacing between the plates is at least 300 um;

wherein another of the configurations is a closed configuration which isconfigured after the fluidic sample is deposited on one or both of thesample contact areas in the open configuration; and in the closedconfiguration: at least part of the sample is confined by the two platesinto a layer, wherein the average sample thickness is 200 um or less;and

wherein, in some embodiments, the heating layer and cooling layer arethe same material layer that has a heating zone and a cooling zone, andwherein the heating zone and cooling zone can have the same area ordifferent areas.

AAA-1.4 A device for rapidly changing the temperature of a fluidicsample, comprising:

a first plate (10), a second plate (20), spacers, a heating layer(112-1), and a cooling layer (112-2), wherein:

the first and second plates are movable relative to each other intodifferent configurations;

each of the first plate and the second plate has, on its respectiveinner surface, a sample contact area for contacting a fluidic sample;wherein the sample contact areas face each other, are separated by anaverage separation distance of 200 um or less between them, and arecapable of contacting the sample and sandwiching the sample betweenthem;

one or both of the plates comprise the spacers and the spacers are fixedon the inner surface of a respective plate;

the spacers have a predetermined substantially uniform height that isequal to or less than 200 microns, and the inter-spacer-distance ispredetermined;

the heating layer is:

positioned on the inner surface, the outer surface, or inside of one ofthe plates, and

configured to heat a relevant volume of the sample, wherein the relevantvolume of the sample is a portion or an entirety of the sample that isbeing heated to a desired temperature; and

the cooling layer is:

positioned on the inner surface, the outer surface, or inside of one ofthe plates; and

configured to cool the relevant sample volume; and

comprises a layer of material that that has a thermal conductivity tothermal capacity ratio of 0.6 cm²/sec or larger;

wherein the distance between the cooling layer and a surface of therelevant sample volume is zero or less than a distance that isconfigured to make the thermal conductance per unit area between thecooling layer and the surface of the relevant sample volume equal to 70W/(m²·K) or larger;

wherein one of the configurations is an open configuration, in which:the two plates are partially or completely separated apart, the spacingbetween the plates is not regulated by the spacers, and the sample isdeposited on one or both of the plates; and

wherein another of the configurations is a closed configuration which isconfigured after the sample is deposited in the open configuration; andin the closed configuration: at least part of the sample is compressedby the two plates into a layer of highly uniform thickness, wherein theuniform thickness of the layer is confined by the sample contactsurfaces of the plates and is regulated by the plates and the spacers;and

wherein, in some embodiments, the heating layer and cooling layer arethe same material layer that has a heating zone and cooling zone, andwherein the heating zone and cooling zone can have the same area ordifferent areas.

AAA-1.5 A device for rapidly changing the temperature of a fluidicsample, comprising:

a first plate (10), a second plate (20), and a heating/cooling layer(112), wherein:

the first plate (10) and the second plate (20) face each other, and areseparated by a distance from each other;

each of the plates has, on its respective inner surface (11, 21), asample contact area for contacting a fluidic sample; wherein the samplecontact areas are facing each other, are in contact with the sample,sandwich a sample between them, and have an average separation distance(102) from each other,

the heating/cooling layer (112) is on the outer surface (22) of thesecond plate (20); and

the heating/cooling layer is configured to comprise a heating zone and acooling zone; wherein the heat zone is configured to heat the fluidicsample, the cooling zone is configured to cool the sample significantlyby thermal radiative cooling;

wherein the heating zone is configured to receive a heating energy froma heating source and to have an area smaller than the total area of theheating/cooling layer; and

wherein at least a part of a heating zone of the heating layer overlapswith the sample area.

AAA-1.6 A device for rapidly changing the temperature of a fluidicsample, comprising:

a first plate (10), a second plate (20), and a heating/cooling layer(112), wherein: each of the first plate (10) and the second plate (20)has, on its respective inner surface (11, 21), a sample contact area forcontacting a fluidic sample; wherein the sample contact areas are facingeach other, are separated by an average separation distance (102) fromeach other, and are capable of contacting the sample and sandwiching thesample between them;

the heating/cooling layer (112) has a thermal conductivity of 50 W/(m·K)or larger and is on the outer surface (22), on the inner surface, orinside of the second plate (20); and

the heating/cooling layer is configured to comprise a heating zone and acooling zone; wherein the heating zone is configured to heat a portionof the sample and have an area smaller than the total area of theheating/cooling layer, and wherein the cooling zone is configured tocool the sample;

wherein the heating zone, the second plate, and the portion of thesample are configured to have a scaled thermal conduction ratio (STCratio) of 2 or larger;

wherein the heating zone is configured to receive a heating energy froma heating source; and wherein at least a part of the heating zone of theheating layer overlaps with the sample area.

AAA-1.7 A device for rapidly changing the temperature of a fluidicsample, comprising:

a first plate (10), a second plate (20), and a heating/cooling layer(112), wherein:

each of the plates has, on its respective inner surface (11, 21), asample contact area for contacting a fluidic sample; wherein the samplecontact areas are facing each other, are in contact with the sample,sandwich the sample between them, and have an average separationdistance (102) from each other;

the heating/cooling layer (112) has a thermal conductivity of 50 W/(m·K)or larger and is on the outer surface (22), on the inner surface, orinside of the second plate (20); and

the heating/cooling layer is configured to comprise a heating zone and acooling zone; wherein the heating zone is configured to heat a portionof the sample and have an area smaller than the total area of theheating/cooling layer, and wherein the cooling zone is configured tocool the sample;

wherein the heating zone, the second plate, and the portion of thesample are configured to have a scaled thermal conduction ratio (STCratio) of 2 or larger;

wherein the heating/cooling layer has a thermal conductivity multiplyingits thickness in the range of 6×10⁻⁵ W/K to 3×10⁻⁴ W/K.

wherein the heating zone is configured to receive a heating energy froma heating source; and

wherein at least a part of the heating zone of the heating layeroverlaps with the sample area.

AAA-2.1. The device of any prior embodiments, wherein the heating layeris configured to be heated by a heating source.

AAA-2.2. The device of any prior embodiments, wherein the heating layeris the same layer as the cooling layer, and the same layer comprises aheating zone area and a cooling zone area.

AAA-2.3. The device of any prior embodiments, wherein the heating layer(i.e. the heating zone) has an area smaller than the cooling layer (i.e.cooling zone).

AAA-2.4. The device of any prior embodiments, wherein the heating layer(i.e., the heating zone) has an area that is about 1/100, 1/50, 1/20,1/10, 1/8, 1/6, 1/5, 1/4, 1/3, 1/2, 2/3, 3/4 or 5/6 of the cooling layer(i.e. cooling zone) area, or in a range between any of the two values.

AAA-2.5. The device of any prior embodiments, wherein the distancebetween the cooling layer and a surface of the relevant sample volume iszero or less than a distance that is configured to make the thermalconductance per unit area between the cooling layer and the surface ofthe relevant sample volume equal to 150 W/(m²·K) or larger.

AAA-2.6. The device of any prior embodiments, wherein the heating layercomprises metallic plasmonic materials, metamaterials, black silicon,graphite, carbon nanotube, silicon sandwich, graphene, or superlattice,or a combination thereof.

AAA-2.7. The device of any prior embodiments, wherein the heating layercomprises Al, Ag, or Au, with or without a paint layer.

AAA-2.8. The device of any prior embodiments, wherein the heating layerhas a thermal conductance per unit area that is equal to or larger than1000 W/(m²·K), 2000 W/(m²·K), 3000 W/(m²·K), 4000 W/(m²·K), 5000W/(m²·K), 7000 W/(m²·K), 10000 W/(m²·K), 20000 W/(m²·K), 50000 W/(m²·K),50000 W/(m²·K), 100000 W/(m²·K), or in range between any of the twovalues.

AAA-2.9. The device of any prior embodiments, wherein the heating layerhas a thermal conductance per unit area that is in a range of 1000W/(m²·K) to 2000 W/(m²·K), 2000 W/(m²·K) to 4000 W/(m²·K), 4000 W/(m²·K)to 10,000 W/(m²·K), or 10000 W/(m²·K) to 100000 W/(m²·K).

AAA3.1 The device of any prior embodiments, wherein the cooling layerhas a thermal conductance per unit area that is equal to or larger than1000 W/(m²·K), 2000 W/(m²·K), 3000 W/(m²·K), 4000 W/(m²·K), 5000W/(m²·K), 7000 W/(m²·K), 10000 W/(m²·K), 20000 W/(m²·K), 50000 W/(m²·K),50000 W/(m²·K), 100000 W/(m²·K), or in range between any of the twovalues.

AAA-3.2. The device of any prior embodiments, wherein the cooling layerhas a thermal conductance per unit area that is in a range of 1000W/(m²·K) to 2000 W/(m²·K), 2,000 W/(m²·K) to 4,000 W/(m²·K), 4,000W/(m²·K) to 10,000 W/(m²·K), or 10,000 W/(m²·K) to 100,000 W/(m²·K).

AAA-3.3 The device of any prior embodiments, wherein the cooling layercools the relevant sample primarily by thermal radiative cooling.

AAA-3.4 The device of any prior embodiments, wherein the cooling of therelevant sample through thermal radiative cooling is larger than thecooling through thermal conduction cooling in the direction lateral tothe plates.

AAA-3.5 The device of any prior embodiments, wherein the cooling of thesample through thermal radiative cooling is at least 1.2 times, 1.5times, 2 times, 5 times, 10 times, times, 50 times, 100 times, 200times, 500 times, or 1000 times larger than the cooling through thermalconduction cooling, or in a range between any of the two values.

AAA4.1 The device of any prior embodiments, wherein the heating layer orthe cooling layer has a thickness that is about 0.1 um, 0.2 um, 0.5 um,1 um, 2 um, 5 um, 10 um, 20 um, 30 um, 40 um, 50 um, 100 um, 200 um, 500um, 1 mm, 2 mm, 5 mm, 10 mm, 20 mm, or 50 mm, or in a range between anyof the two values.

AAA4.2 The device of any prior embodiments, wherein the heating layer orthe cooling layer has an area that is less than 0.01 mm², 0.02 mm², 0.05mm², 0.1 mm², 0.2 mm², 0.5 mm², 1 mm², 2 mm², 5 mm², 10 mm², 20 mm², 50mm², 100 mm², 200 mm², 500 mm², or 1000 mm², or in a range between anyof the two values.

AAA4.3 The device of any prior embodiments, wherein the heating layer orthe cooling layer has an area dimension that is about 1 mm, 2 mm, 3 mm,5 mm, 6 mm, 7 mm, 8 mm, 9 mm 10 mm, 12 mm, or 15 mm, or in a rangebetween any two values.

AAA4.4 The device of any prior embodiments, wherein the heating layer orthe cooling layer comprises metallic plasmonic materials, metamaterials,black silicon, graphite, carbon nanotube, silicon sandwich, graphene, orsuperlattice, or a combination thereof.

AAA5.1 The device of any prior embodiments, wherein the heating layerand the cooling layer are structurally separate layers, the heatinglayer has a heating zone, and the cooling layer has a cooling zone.

AAA6.1 The device of any prior embodiments, wherein the ratio of thecooling zone area to the heating zone area is larger than 1, 1.5, 2,2.5, 3, 5, 10, 15, 20, 25, 50, 75, 100, 200, 500, or 1000, or in a rangebetween any of the two values.

AAA6.2 The device of any prior embodiments, wherein the cooling zonearea is larger than the lateral area of the relevant sample volume by afactor that is equal to or large than 1.2 times, 1.5 times, 2 times, 5times, 10 times, 20 times, 50 times, 100 times, 200 times, 500 times, or1000 times larger than the cooling through thermal conduction cooling,or in a range between any of the two values.

AAA6.3 The device of any prior embodiments, wherein the cooling of thedevice has a thermal radiation cooling that, during a thermal cycling,is equal to or larger than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, or 99% of the total cooling or in a range between any of the twovalues, wherein the total cooling is the sum of radiative cooling andconductive cooling.

AAA6.4 The device of any prior embodiments, wherein the cooling of thedevice by thermal radiation through a high K cooling layer, during athermal cycling, is equal to or larger than. 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 99% of the total cooling or in a range between any ofthe two values, wherein the total cooling is the sum of radiativecooling and conductive cooling.

AAA7.1 The device of any prior embodiments, wherein at least one of theplates is flexible.

AAA8.1 The device of any prior embodiments, wherein the device comprisesspacers that regulate the thickness of the sample when the sample isconfined by the two plates into a thin layer.

AAA8.2 The device of any prior embodiments, wherein the spacers has aninter-spacer-distance (ISD), and wherein the fourth power of theinter-spacer-distance (ISD) divided by the thickness (h) and the Young'smodulus (E) of the flexible plate (ISD4/(hE)) is 5×10⁶ um³/GPa or less.

AAA8.3. The device of any prior embodiments, wherein the spacers has acontact filling factor, wherein the product of the contact fillingfactor and the Young's modulus of the spacers is 2 MPa or larger, andwherein the contact filling factor is, in the sample contact area, theratio of the contact area between spacer and the plate to the totalplate area.

AAA8.4. The device of any prior embodiments, wherein the spacers are inthe sample contact area.

AAA8.5. The device of any prior embodiments, wherein the spacers have ashape of the pillars with substantially flat top.

AAA8.6. The device of any prior embodiments, wherein the spacers arefixed on either one or both of the plates.

AAA8.7. The device of any prior embodiments, wherein the spacers have auniform height.

AAA8.8. The device of any prior embodiments, wherein the thickness ofthe sample is the same as the height of the spacers.

AAA9.1 The device of any prior embodiments, wherein the heating layerand/or the cooling layer is on the inner surface of one of the plates.

AAA9.2 The device of any prior embodiments, wherein the heating layerand/or the cooling layer is on the outer surface of one of the plates.

AAA9.3 The device of any prior embodiments, wherein the heating layerand the cooling layer are separate, the heating layer is on the outersurface of one of the plates, and the cooling layer is on the outersurface of the other plate.

AAA9.4 The device of any prior embodiments, wherein the heating layerand the cooling layer are separate, the heating layer is on the innersurface of one of the plates, and the cooling layer is on the innersurface of the other plate.

AAA9.5 The device of any prior embodiments, wherein the heating layerand the cooling layer are separate, the heating layer is on the inner orouter surface of one of the plates, and the cooling layer is on theinner or outer surface of the other plate.

AAA9.6 The device of any prior embodiments, wherein the heating layerand the cooling layer are inside one or both of the plates.

AAA9.7 The device of any prior embodiments, wherein the heating zone andthe cooling zone are partly overlapping on the heating and/or coolinglayer.

AAA10.1 The device of any prior embodiments, wherein the first plate orthe second plate has a thickness that is less than 10 nm, 100 nm, 200nm, 500 nm, 1000 nm, 2 μm (micron), 5 μm, 10 μm, 20 μm, 50 μm, 100 μm,150 μm, 200 μm, 300 μm, 500 μm, 800 μm, 1 mm (millimeter), 2 mm, 3 mm, 5mm, 10 mm, 20 mm, 50 mm, 100 mm, 500 mm, or in a range between any twoof these values.

AAA10.2 The device of any prior embodiments, wherein the first plate orthe second plate has an lateral area that is less than 1 mm² (squaremillimeter), 10 mm², 25 mm², 50 mm², 75 mm², 1 cm² (square centimeter),2 cm², 3 cm², 4 cm², 5 cm², 10 cm², 100 cm², 500 cm², 1000 cm², 5000cm², 10,000 cm², 10,000 cm², or in a range between any two of thesevalues.

AAA10.3.1 The device of any prior embodiments, wherein the first plateor the second plate comprises PMMA.

AAA10.4 The device of any prior embodiments, wherein the plates arethermal-isolated from a structure that accommodate the plates.

AAA11.1 The device of any prior embodiments, wherein the relevant samplehas a volume that is about 0.01 ul, 0.02 ul, 0.05 ul, 0.1 ul, 0.2 ul,0.5 ul, 1 ul, 2 ul, 5 ul, 10 ul, 20 ul, 50 ul, 100 ul, 200 ul, 500 ul, 1ml, 2 ml, 5 ml, 10 ml, 20 ml, or 50 ml, or in a range between any of thetwo values.

AAA11.2 The device of any prior embodiments, wherein ratio of thelateral average dimension of the relevant sample area to the samplethickness is greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50,60, 70, 80, 90, 100, 200, 500, 1000, 2000, 5000, 100,000, or in a rangebetween any of the two values.

AAA12.1 The device of any prior embodiments, wherein the plates areconfigured to be operable directly by human hands.

AAA12.2 The device of any prior embodiments, wherein the plates areconfigured to be compressed directly by human hands with impreciseforce, which is neither set to a precise level nor substantiallyuniform.

AAA12.3 The device of any prior embodiments, further comprising a hinge,which connects the first plate and the second plate and allows the twoplates to pivot against each other into different configurations.

AAA12.4 The device of any prior embodiments, wherein at least one of theplates comprises one or more open notches on an edge or corners of theplate, and the notch(es) facilitates changing the plates betweendifferent configurations.

AAA12.5 The device of any prior embodiments, wherein at least one of theplates comprises one or more open notches on an edge or corners of theplate, and the notch(es) facilitates changing the plates from aconfiguration that is near or at closed configuration to an openconfiguration.

AAA13.1. A sample cartridge, comprising the device of any priorembodiments, and a sample support that is configured to support thedevice.

AAA13.2. The sample cartridge of any prior embodiments, wherein thesample support comprises one or more apertures that allow energy toreach the heating layer.

AAA14.1 An apparatus for rapidly changing temperature of a fluidicsample, comprising:

the device of any prior embodiments;

a heating source that is configured to supply energy to the device.

AAA14.2 The apparatus of any prior embodiments, wherein the heatingsource is configured to radiate electromagnetic waves in a range ofwavelength that the heating/cooling layer has an absorption coefficientof 50% or higher.

AAA14.3. The apparatus of any prior embodiments, wherein the heatingsource comprises one or an array of light-emitting diodes (LEDs), one oran array of lasers, one or an array of lamps, or a combination ofthereof.

AAA14.4. The apparatus of any prior embodiments, wherein the heatingsource comprises halogen lamp, halogen lamp with reflector, LED withfocusing lens, laser with focusing lens, halogen lamp with couplingoptical fiber, LED with coupling optical fiber, laser with couplingoptical fiber.

AAA14.5 The apparatus of any prior embodiments, further comprising anoptical pipe between the heating source and the device, wherein theoptical pipe is configured to guide the energy from the heating sourceto the heating layer.

AAA15.1 An apparatus for rapidly changing temperature of a fluidicsample, comprising:

the device of any prior embodiments; and

an adaptor that is configured to accommodate the device.

AAA15.2 The apparatus of any prior embodiments, wherein the adaptorcomprises a sample slot that is configured to accommodate the device andposition the device to receive the electromagnetic waves from theheating source.

AAA15.3 The apparatus of any prior embodiments, wherein adaptorcomprises a slider that is configured to allow the device to slide intothe sample slot.

AAA16.1 An apparatus for rapidly changing temperature of a fluidicsample, comprising:

the device of any prior embodiments;

a heating source that is configured to supply energy the device; and

a control unit that is configured to control the heating unit.

AAA16.2 The apparatus of any prior embodiments, wherein the control unitis configured to control electromagnetic waves from the heating source.

AAA16.3 The apparatus of any prior embodiments, wherein the control unitis configured to control the presence, intensity, wavelength, frequency,and/or angle of the electromagnetic waves.

AAA16.2 The apparatus of any prior embodiments, wherein the control unitcomprises a temperature sensor that is configured to detect thetemperature of the sample.

A16.2.1 The apparatus of any prior embodiments, wherein the control unitcontrols the energy supplied by the heating source based on thetemperature detected by the temperature sensor.

AAA17.1. A system for rapidly changing temperature of a fluidic sample,comprising:

a device of any prior embodiments;

a heating source that is configured to emit electromagnetic waves thatcan be received by the device; and

a control unit, which is configured to control heating and cooling ofthe sample, at least in part by changing the electromagnetic waves fromthe heating source.

AAA17.2 The system of any prior embodiments, further comprising anadaptor that is configured to accommodate the device.

AAA17.3 The system of any prior embodiments, further comprising anoptical pipe that is configured to guide the electromagnetic waves fromthe heating source to the device.

AAA17.4 The system of any prior embodiments, further comprising a signalsensor that is configured to detect a signal from the sample.

AAA17.4.1. The system of any prior embodiments, wherein the signalsensor is an optical sensor that is configured to image the fluidicsample.

AAA18.1 A kit for rapidly changing temperature of a fluidic sample,comprising: a device of any prior embodiments; and reagents thatconfigured to facilitate a chemical/biological reaction.

AAA18.2 The kit of any prior embodiments, wherein the reagents areconfigured for nucleic acid amplification:

AAA18.3 The kit of any prior embodiments, wherein the reagents comprisesa pre-mixed polymerase chain reaction (PCR) medium:

AAA18.4 The kit of any prior embodiments, wherein the reagents areconfigured to detect nucleic acids by amplifying (generating numerouscopies of) the target molecules in samples, wherein target moleculerefers to a sequence, or partial sequence, of nucleic acid of interest.

AAA18.5 The kit of any prior embodiments, wherein the reagents comprise:primers, deoxynucleotides (dNTPs), bivalent cations (e.g. Mg²⁺),monovalent cation (e.g. K⁺), buffer solutions, enzymes, or reporters, orany combination or mixture thereof.

AAA18.6 The kit of any prior embodiments, wherein the reagents areeither in the dry form on the inner surface of the first or the secondplate or both, or in a liquid form encased in, embedded in, orsurrounded by, a material that melts with increasing temperatures, suchas, for example, paraffin.

AAA18.7 The kit of any prior embodiments, wherein the reagents compriseDNA-dependent polymerase, or RNA-dependent DNA polymerase, orDNA-dependent RNA polymerase.

AAA18.8 The kit of any prior embodiments, wherein the reagents comprise“reporters” that refer to any tag, label, or dye that can bind to, orintercalate within, the nucleic acid molecule or be activated bybyproducts of the amplification process to enable visualization of thenucleic acid molecule or the amplification process.

AAA18.9 The kit of any prior embodiments, wherein the reagents comprisecell lysing reagent, which is configured to facilitate breaking downcellular structures.

AAA19.1 The device, apparatus, system, and/or kit of any priorembodiments, wherein the heating layer and/or the cooling layer areattached to the first plate and/or the second plate by e-beamevaporation.

AAA19.2. The device, apparatus, system, and/or kit of any priorembodiments, wherein the heating layer and/or the cooling layer comprisegold and the gold is attached to the first plate and/or the second plateby e-beam evaporation.

AA1. A device for rapidly changing a fluidic sample temperature,comprising:

a first plate, a second plate, and a heating/cooling layer, wherein:

the plates are movable relative to each other into differentconfigurations;

each of the plates has, on its respective inner surface, a samplecontact area for contacting a fluidic sample, and

the heating/cooling layer is configured to heat the fluidic sample;

wherein the heating/cooling layer is (a) on (either the inner or outersurface) or inside one of the plates, and (b) capable of being heated bya heating source, wherein the heating source delivers heat energy to theheating/cooling layer optically, electrically, by radio frequency (RF)radiation, or a combination thereof;

wherein at least a part of a heating area of the heating/cooling layeroverlaps with the sample area,

wherein one of the configurations is an open configuration, in which:the two plates are partially or completely separated apart and theaverage spacing between the plates is at least 300 um; and

wherein another of the configurations is a closed configuration which isconfigured after the fluidic sample is deposited on one or both of thesample contact areas in the open configuration; and in the closedconfiguration: at least part of the sample is compressed by the twoplates into a layer, wherein the average sample thickness is 200 um orless.

AA2.1 A device for rapidly changing temperature of a fluidic sample,comprising:

a sample holder and a heating/cooling layer, wherein:

the sample holder comprises a first plate and a second plate, whereineach of the plates comprises, on its respective surface, a samplecontact area for contacting the fluidic sample;

the first plate and the second plate are configured to confine thefluidic sample into a layer of highly uniform thickness of 0.1-200 umand substantially stagnant relative to the plates; and

the heating/cooling layer: (1) has a thickness of less than 1 mm, (2)has an area that is substantially less than the area of either the firstor the second plate, and (3) is configured to convert energy fromelectromagnetic waves into heat to raise the temperature of at leastpart of the fluidic sample in the layer of uniform thickness.

AA2.2 A device for rapidly changing temperature of a fluidic sample,comprising:

a sample holder and a heating/cooling layer, wherein:

the sample holder comprises a first plate and a second plate, whereineach of the plates comprises, on its respective surface, a samplecontact area for contacting the fluidic sample;

the first plate and the second plate are configured to confine at leastpart of the sample into a layer of highly uniform thickness of 0.1-200um and substantially stagnant relative to the plates,

the first plate has a thickness of 500 um or less, and the second platehas a thickness of 5 mm or less; and

the heating/cooling layer has a thickness of less than 1 mm and an areaof less than 100 mm² and is configured to convert energy fromelectromagnetic waves into heat to raise the temperature of the at leastpart of the fluidic sample in the layer of uniform thickness.

AA2.3 A device for rapidly changing temperature of a fluidic sample,comprising:

a sample holder and a heating/cooling layer, wherein:

the sample holder comprises a first plate and a second plate, whereineach of the plates comprises, on its respective surface, a samplecontact area for contacting the fluidic sample;

the first plate and the second plate are configured to confine at leastpart of the sample into a layer of highly uniform thickness of 0.1-200um and substantially stagnant relative to the plates,

the first plate has a thickness of 500 um or less, and the second platehas a thickness of 5 mm or less; and

the heating/cooling layer: (1) has a thickness of less than 1 mm, (2)has an area of less than 100 mm² that is substantially less than thearea of either the first or the second plate, and (3) is configured toconvert energy from electromagnetic waves into heat to raise thetemperature of at least part of the fluidic sample in the layer ofuniform thickness.

AA3 A device for rapidly changing temperature of a fluidic sample,comprising:

a sample holder and a heating/cooling layer, wherein:

the sample holder comprises a first plate and a second plate, whereineach of the plates comprises, on its respective surface, a samplecontact area for contacting the fluidic sample;

the first plate and the second plate are configured to confine at leastpart of the sample into a layer of highly uniform thickness of 500 um orless and substantially stagnant relative to the plates,

the first plate is in contact with the heating/cooling layer and has athickness of 1 um or less, and the second plate is not in contact withthe heating/cooling layer and has a thickness of 5 mm or less; and

the heating/cooling layer is configured to convert energy fromelectromagnetic waves into heat to raise the temperature of the at leastpart of the fluidic sample in the layer of uniform thickness, has anabsorption coefficient of 50% or higher, and has a thickness of lessthan 3 mm.

AA4 A device for rapidly changing temperature of a fluidic sample,comprising:

a sample holder and a heating/cooling layer, wherein:

the sample holder comprises a first plate and a second plate, whereineach of the plates comprises, on its respective surface, a samplecontact area for contacting the fluidic sample;

the first plate and the second plate are configured to confine at leastpart of the sample into a layer of highly uniform thickness of 500 um orless and substantially stagnant relative to the plates,

the first plate is in contact with the heating/cooling layer and has athickness of 1 um or less, and the second plate is not in contact withthe heating/cooling layer and has a thickness of 0.1-2 mm; and

the heating/cooling layer is configured to convert energy fromelectromagnetic waves into heat to raise the temperature of the at leastpart of the fluidic sample in the layer of uniform thickness, has anabsorption coefficient of 60% or higher, and has a thickness of lessthan 2 mm.

AA6.1 The device of any prior embodiments, wherein the heating/coolinglayer is on the inner surface of one of the plates.

AA6.2 The device of any prior embodiments, wherein the heating/coolinglayer is on the outer surface of one of the plates.

AA6.3 The device of any prior embodiments, wherein the heating/coolinglayer inside one of plates.

AA6.4 The device of any prior embodiments, wherein the heating/coolinglayer is in contact with at least one of the plates.

AA6.5 The device of any prior embodiments, wherein the heating/coolinglayer is not in contact with any of the plates.

AA6.6 The device of any prior embodiments, wherein the heating/coolinglayer is in contact with the sample when the plates are in the closedconfiguration.

AA7. The device of any prior embodiments, wherein the heating/coolinglayer is made from a single material or compound materials.

AA7.1 The device of any prior embodiments, wherein the heating/coolinglayer comprises semiconductors or metallic materials with high absorbingsurfaces.

AA7.2 The device of any prior embodiments, wherein the heating/coolinglayer comprises Silicon, Ge, InP, GaAs, CdTe, CdS, aSi, metal includingAu, Al, Ag, Ti, carbon coated Al, black painted Al, carbon (graphene,nanotube, nanowire) or a combination thereof.

AA7.3 The device of any prior embodiments, wherein the heating/coolinglayer is acting as the fast heating conductive layer comprises Silicon,Ge, InP, GaAs, CdTe, CdS, aSi, metal including Au, Al, Ag, Ti, carboncoated Al, black painted Al, carbon (graphene, nanotube, nanowire) or acombination thereof.

AA8 The device of any prior embodiments, wherein the part of the heatingarea that overlaps the sample area is less than 1%, 5%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the sample area, or in arange between any of the two values.

AA8.1 The device of any prior embodiments, wherein the part of theheating area that overlaps the sample area is less than 0.1 mm², 0.5mm², 1 mm², 5 mm², 10 mm², 25 mm², 50 mm², 75 mm², 1 cm² (squarecentimeter), 2 cm², 3 cm², 4 cm², 5 cm², 10 cm², or in a range betweenany of the two values.

AA9. The device of any prior embodiments, wherein the absorptioncoefficient of the heating/cooling layer is more than 30%, 40%, 50%,60%, 70%, 80%, 90%, or in a range between any of the two values.

AA9.1. The device of any prior embodiments, wherein the absorptioncoefficient of the heating/cooling layer is more than 60%, 70%, 80%,90%, or in a range between any of the two values.

AA9.2. The device of any prior embodiments, wherein the absorptioncoefficient of the heating/cooling layer is more than 60%.

AA10. The device of any prior embodiments, wherein the heating/coolinglayer has an absorption wavelength range that is 100 nm to 300 nm, 400nm to 700 nm (visible range), 700 nm to 1000 nm (IR range), 1 um to 10um, 10 um to 100 um, or in a range between any of the two values.

AA11. The device of any prior embodiments, wherein the heating/coolinglayer has a thickness equal to or less than 3 mm, 2 mm, 1 mm, 750 um,500 um, 250 um, 100 um, 50 un, 25 um, 10 um, 500 nm, 200 nm, 100 nm, or50 nm, or in a range between any of the two values.

AA12. The device of any prior embodiments, wherein the heating/coolinglayer has an area of 0.1 mm² or less, 1 mm² or less, 10 mm² or less, 25mm² or less, 50 mm² or less, 75 mm² or less, 1 cm² (square centimeter)or less, 2 cm² or less, 3 cm² or less, 4 cm² or less, 5 cm² or less, 10cm² or less, or in a range between any of the two values.

AA13. The device of any prior embodiments, wherein the first plate has athickness equal to or less than 500 um, 200 um, 100 um, 50 um, 25 um, 10um, 5 um, 2.5 um, 1 um, 500 nm, 400 nm, 300 nm, 200 nm, or 100 nm, or ina range between any of the two values.

AA13.1. The device of any prior embodiments, wherein the first plate hasa thickness equal of 10-200 um.

AA14. The device of any prior embodiments, wherein the second plate hasa thickness equal to or less than 5 mm, 4 mm, 3 mm, 2 mm, 1 mm, 750 um,500 um, 250 um, 100 um, 75 um, 50 um, or 25 um, or in a range betweenany of the two values.

AA14.1. The device of any prior embodiments, wherein the second platehas a thickness equal of 10-1000 um.

AA15. The device of any prior embodiments, wherein the sample layer hasa highly uniform thickness.

AA15.1 The device of any prior embodiments, wherein the sample layer hasa thickness of equal to or less than 100 um, 50 um, 20 um, 10 um, 5 um,1 um, 500 nm, 400 nm, 300 nm, 200 nm, or 100 nm, or in a range betweenany of the two values.

AA15.2. The device of any prior embodiments, wherein the sample layerhas a thickness of 1-100 um.

AA16. The device of any prior embodiments, wherein the area of at leastone of the plate is 1 mm² or less, 10 mm² or less, 25 mm² or less, 50mm² or less, 75 mm² or less, 1 cm² (square centimeter) or less, 2 cm² orless, 3 cm² or less, 4 cm² or less, 5 cm² or less, 10 cm² or less, 100cm² or less, 500 cm² or less, 1000 cm² or less, 5000 cm² or less, 10,000cm² or less, 10,000 cm² or less, or in a range between any two of thesevalues.

AA17.1 The device of any prior embodiments, wherein the area of at leastone of the plates is in the range of 500 to 1,000 mm²; or around 750mm².

AA18. The device of any prior embodiments, further comprising spacersthat are configured to regulate the thickness of the sample layer.

AA18.1 The device of any prior embodiments, wherein the spacers arefixed on either one or both of the plates.

AA18.2 The device of any prior embodiments, wherein the spacers arefixed on the inner surface of either one or both of the plates.

AA18.3 The device of any prior embodiments, wherein the spacers have auniform height.

AA18.4 The device of any prior embodiments, wherein at least one of thespacers is inside the sample contact area.

AA18.5 The device of any prior embodiments, wherein the thickness of thesample layer is the same as the height of the spacers.

AA19 The device any prior embodiments, wherein one or both plates areflexible.

AA20. The device of any prior embodiments, further comprising sealingstructures that are attached to either one or both of the contact andsecond plates, wherein the sealing structures are configured to limitthe evaporation of liquid inside the device.

AA21. The device of any prior embodiments, further comprising a clampingstructure that is attached to either one or both of the first and secondplates, wherein the clamp structure is configured to hold the device andregulate the thickness of the sample layer during the heating of thedevice.

AA22. The device of any prior embodiments, wherein the second plate istransparent for an electromagnetic wave from the sample.

AA23. The device of any prior embodiments, wherein the sample holder andthe heating/cooling layer are connected by a thermal coupler.

AA24. The device of any prior embodiments, wherein the areas of the atleast part of the sample and the heating/cooling layer are substantiallylarger than the uniform thickness.

AA25. The device of any prior embodiments, wherein the heating/coolinglayer is configured to absorb electromagnetic waves selected from thegroup consisting of: radio waves, microwaves, infrared waves, visiblelight, ultraviolet waves, X-rays, gamma rays, and thermal radiation.

AA26. The device of any prior embodiments, wherein the sample is apre-mixed polymerase chain reaction (PCR) medium.

AA27. The device of any prior embodiments, wherein the sample layer islaterally sealed to reduce sample evaporation.

AA28. The device of any prior embodiments, wherein the area of theradiation is smaller than the area of radiation absorption pad; The areaof the radiation absorption pad is less than the area of sample liquidarea; The area of sample liquid area is less than the first and secondplate size.

AA29. The device of any prior embodiments, wherein the fluidic samplecomprises a processed or unprocessed bodily fluid.

AA30. The device of any prior embodiments, wherein the fluidic samplecomprises amniotic fluid, aqueous humour, vitreous humour, blood (e.g.,whole blood, fractionated blood, plasma, serum, etc.), breast milk,cerebrospinal fluid (CSF), cerumen (earwax), chyle, chime, endolymph,perilymph, feces, gastric acid, gastric juice, lymph, mucus (includingnasal drainage and phlegm), pericardial fluid, peritoneal fluid, pleuralfluid, pus, rheum, saliva, sebum (skin oil), semen, sputum, sweat,synovial fluid, tears, vomit, urine and exhaled condensate. In someembodiments, the sample comprises a human body fluid. In someembodiments, the sample comprises at least one of cells, tissues, bodilyfluids, stool, amniotic fluid, aqueous humour, vitreous humour, blood,whole blood, fractionated blood, plasma, serum, breast milk,cerebrospinal fluid, cerumen, chyle, chime, endolymph, perilymph, feces,gastric acid, gastric juice, lymph, mucus, nasal drainage, phlegm,pericardial fluid, peritoneal fluid, pleural fluid, pus, rheum, saliva,sebum, semen, sputum, sweat, synovial fluid, tears, vomit, urine, orexhaled condensate, or a mixture thereof.

AA31. The device of any prior embodiments, wherein the fluidic samplecomprises nucleic acids or proteins, or a mixture thereof.

AA32. The device of any prior embodiments, wherein the fluidic samplecomprises DNA or RNA, or a mixture thereof.

Apparatus with Heating Source

BB1. An apparatus for rapidly changing temperature of a fluidic sample,comprising:

a holder that can hold a device of any AA embodiments; and

a heating source that is configured to supply energy to theheating/cooling layer; and

a controller that is configured to control the heating source.

BB1.1 The apparatus of any prior BB embodiments, wherein the heatingsource is configured to radiate electromagnetic waves in a range ofwavelength that the heating/cooling layer has an absorption coefficientof 50% or higher.

BB2. The apparatus of any prior BB embodiments, wherein the heatingsource comprises one or an array of light-emitting diodes (LEDs), one oran array of lasers, one or an array of lamps, or a combination ofthereof.

BB2.1. The apparatus of any prior BB embodiments, wherein the heatingsource comprises halogen lamp, halogen lamp with reflector, LED withfocusing lens, laser with focusing lens, halogen lamp with couplingoptical fiber, LED with coupling optical fiber, laser with couplingoptical fiber.

BB3. The apparatus of any prior BB embodiments, wherein the wavelengthis 50 nm, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450nm, 500 nm, 550 nm, 600 nm, 650 nm, 700 nm, 750 nm, 800 nm, 850 nm, 900nm, 950 nm, 1 um, 10 um, 25 um, 50 um, 75 um, or 100 um, or in a rangebetween any of the two values.

BB3.1 The apparatus of any prior BB embodiments, wherein the wavelengthof the electromagnetic waves is 100 nm to 300 nm, 400 nm to 700 nm(visible range), 700 nm to 1,000 nm (IR range), 1 um to 10 um, 10 um to100 um, or in a range between any of the two values.

BB4. The apparatus of any prior BB embodiments, further comprising aheat sink that is configured to absorb at least part of the heatradiated from the sample holder and/or the heating source.

BB4.1. The apparatus of any prior BB embodiments, wherein the heat sinkis chamber that at least partially encloses the device.

BB4.2. The apparatus of any prior BB embodiments, wherein the chambercomprises a lower aperture configured to allow passage ofelectromagnetic waves from the heating source to the heating/coolinglayer, and an upper aperture configured to allow imaging of the sample.

BB5. The apparatus of any prior BB embodiments, wherein the sampleholder is heated optically, electrically, by RF, or a combination ofthereof.

BB6. An apparatus for rapidly changing temperature of a fluidic sample,comprising: a device of any AA embodiments; and a heat sink that isconfigured to absorb at least part of the heat radiated from the sampleholder and/or the heating source.

BB7. The apparatus of any prior BB embodiments, wherein the heat sink isa chamber that at least partially encloses the device, wherein thechamber comprises a radiation aperture configured to allow passage ofelectromagnetic waves from a heating source to the heating/coolinglayer, and an optical aperture configured to allow imaging of thesample.

BB8. The apparatus of any prior BB embodiments, further comprising acooling member attached to the chamber, wherein the cooling member isconfigured to reduce temperature in the chamber.

BB9. The apparatus of embodiment BB7, wherein the cooling member is afan.

BB10. The apparatus of embodiment BB7, wherein the cooling member is aPeltier cooler.

BB11. The apparatus of any BB embodiments, wherein the chamber has anon-reflective inner surface.

BB11.1 The apparatus of any BB embodiments, wherein the chamber has aninner surface made of black metal.

BB12. The apparatus of any BB embodiments, wherein the device issuspended (i.e. has minimum) thermal conduction contact with the chamberwall.

BB13. The apparatus of any BB embodiments, wherein the heat sink isconfigured to connect the sample holder to a mobile device.

BB13.1 The apparatus of embodiment B13, wherein the mobile device is asmartphone comprising a camera.

BB14. The apparatus of any BB embodiments, wherein the heat sinkcomprises optical elements that optimizes capturing images of the samplein the sample card.

CC1. A system for rapidly changing temperature of a fluidic sample,comprising: a device of any AA embodiments or an apparatus of any BBembodiments; and a signal sensor that is configured to senses a signalfrom the sample on the device.

CC2. The system of any prior CC embodiments, wherein the signal sensoris an optical sensor that is configured to image the fluidic sample.

CC2.1 The system of any prior CC embodiments, wherein the optical sensoris a photodetector, camera, or a device capable of capturing images ofthe fluidic sample.

CC3. The system of any prior CC embodiments, wherein the signal sensoris an electrical sensor that is configured to detect electrical signalsfrom the device.

CC4. The system of any prior CC embodiments, wherein the signal sensoris a mechanical sensor that is configured to detect mechanical signalsfrom the device.

CC5. The system of any prior CC embodiments, wherein the signal sensoris configured to monitor the amount of an analyte in the sample.

CC6. The system of any prior CC embodiments, wherein signal sensor isoutside the chamber and receive optical signals from the sample throughan optical aperture on the chamber.

CC7. The system of any CC embodiment, further comprising a thermalcoupler bound to the heating/cooling layer.

CC8. The system of any prior CC embodiments, further comprising athermostat that monitor the temperature of the heating/cooling layer.

CC9. The system of any prior CC embodiments, further comprising atemperature monitoring dye that is configured to facilitate monitoringthe temperature of the sample in the device.

CC9.1. The system of any prior CC embodiments, wherein the temperaturemonitoring dye is in liquid form.

CC9.2. The system of any prior CC embodiments, wherein the temperaturemonitoring dye comprises LDS 688, LDS 698, LDS 950, LD 390, LD 423, LD425, or IR 144, or a combination thereof.

DD1. The device, apparatus, or system of any prior embodiments, wherein:

there are spacers that are fixed on one of both of the plates, whereinat least one of the spacers is in the sample contact area;

the sample layer has a thickness of 0.1-200 um;

the first plate is in contact with the heating/cooling layer and has athickness of 500 um or less, and the second pate is not in contact withthe heating/cooling layer and has a thickness of 5 mm or less; and

the heating/cooling layer: (1) has a thickness of less than 1 mm, (2)has an area of less than 100 mm² that is substantially less than thearea of either the first or the second plate, and (3) is configured toconvert energy from electromagnetic waves into heat to raise thetemperature of at least part of the fluidic sample in the layer ofuniform thickness.

DD2. The device, apparatus, or system of any prior embodiments, wherein:

the heating/cooling layer is on the inner surface of the first plate andin contact with the sample when the plates are in the closedconfiguration;

the heating/cooling layer is made from silicon; and

there is a chamber that encloses the sample holder and the chamber has anon-reflective inner surface.

DD3. The device, apparatus, or system of any prior embodiments, wherein:

there is a heating source that is configured to radiate electromagneticwaves in a range of wavelength that the heating/cooling layer has anabsorption coefficient of 50% or higher;

there is a chamber that comprises a lower aperture configured to allowpassage of electromagnetic waves from the heating source to theheating/cooling layer, and an upper aperture configured to allow imagingof the sample; and

there is an optical sensor that is configured to capture images of thefluidic sample in the sample holder.

EE1.1. A method for rapidly changing temperature of a fluidic sample,comprising:

providing a device that comprises a first plate, a second plate, aheating layer, and a cooling layer, wherein:

each of the plates comprises, on its respective inner surface, a samplecontact area;

the heating layer is positioned on the inner surface, the outer surface,or inside of one of the plates; and is configured to heat a relevantvolume of the sample, wherein the relevant volume of the sample is aportion or an entirety of the sample that is being heated to a desiredtemperature; and

the cooling layer is positioned on the inner surface, the outer surface,or inside of one of the plates; is configured to cool the relevantsample volume; and comprises a layer of material that that has a thermalconductivity to thermal capacity ratio of 0.6 cm²/sec or larger, whereinthe high thermal conductivity to thermal capacity ratio layer has anarea larger than the lateral area of the sample volume;

wherein the distance between the cooling layer and a surface of therelevant sample volume is zero or less than a distance that isconfigured to make the thermal conductance per unit area between thecooling layer and the surface of the relevant sample volume equal to 150W/(m²·K) or larger.

depositing a fluidic sample on one or both of the sample contact areasof the respective plates;

pressing the plates, by hand, to make the sample contact areas face eachother, wherein the plates are separated by an average separationdistance of 200 um or less, sandwiching the sample between them andpressing at least part of the sample into a thin layer:

changing and/or maintaining the temperature of the relevant volume inthe device.

EE1.2. A method for rapidly changing temperature of a fluidic sample,comprising:

providing the device of the SC-A embodiments:

depositing a fluidic sample on one or both of the sample contact areasof the respective plates;

pressing the plates, by hand, to sandwich the sample between them andpressing at least part of the sample into a thin layer:

changing and/or maintaining the temperature of the relevant volume inthe device.

EE1.3. A method for rapidly changing temperature of a fluidic sample,comprising: obtaining the system of the CC embodiments;

depositing the fluidic sample in the sample holder;

pressing the first plate and the second plate to compress at least partof the sample into a layer of uniform thickness; and

changing and maintaining the temperature of the sample layer by changingthe presence, intensity, wavelength, frequency, and/or angle of theelectromagnetic waves from the heating source.

EE2. The method of any prior EE embodiments, wherein changing thetemperature of the sample layer comprises raising the temperature orlowering the temperature.

EE3. The method of any prior EE embodiments, further comprising imagingthe sample layer with the optical sensor.

EE4. The method of any prior EE embodiments, further comprisingmonitoring the temperature of the sample layer and adjusting the step ofchanging and maintaining the temperature of the sample layer.

EE5. The method of any prior EE embodiments, wherein the step ofchanging and maintaining the temperature of the sample layer isconducted according to a pre-determined program.

EE6. The method of any prior EE embodiments, wherein the method iscustomized to facilitate polymerase chain reaction (PCR) assays forchanging temperature of the sample according to a predetermined program

EE7. The method of any prior EE embodiments, further comprisingmonitoring the amount of an analyte in the sample in real time.

FF1. The device, apparatus, system or method of any prior embodiments,wherein the sample comprises nucleic acids.

FF1.1 The device, apparatus, system or method of any prior embodiments,wherein the sample comprises DNA.

FF1.2 The device, apparatus, system or method of any prior embodiments,wherein the sample comprises RNA.

FF1.3 The device, apparatus, system or method of any prior embodiments,wherein the sample comprises DNA or RNA molecule, or a DNA/RNA hybrid,or mixtures of DNA and/or RNA.

FF1.4 The device, apparatus, system or method of any prior embodiments,wherein the sample comprises genomic or chromosomal DNA, plasmid DNA,amplified DNA, cDNA, total RNA, mRNA and small RNA.

FF1.5 The device, apparatus, system or method of any prior embodiments,wherein the sample comprises natural DNA and/or RNA molecule, orsynthetic DNA and/or RNA molecule.

FF1.6 The device, apparatus, system or method of any prior embodiments,wherein the sample comprises cell-free nucleic acids, wherein“cell-free” refers to nucleic acids are not contained in any cellularstructures.

FF1.7 The device, apparatus, system or method of any prior embodiments,wherein the sample comprises nucleic acids are contained within cellularstructures, which include but not limited to human cells, animal cells,plant cells, bacterial cells, fungi cells, and/or viral particles.

FF1.8 The device, apparatus, system or method of any prior embodiments,wherein the sample comprises purified nucleic acids.

FF2. The device, apparatus, system or method of any prior embodiments,wherein the sample comprises proteins and/or lipids.

FF3. The device, apparatus, system or method of any prior embodiments,wherein the sample comprises reagents configured for nucleic acidamplification.

FF3.1. The device, apparatus, system or method of any prior embodiments,wherein the sample comprises a pre-mixed polymerase chain reaction (PCR)medium.

FF3.2. The device, apparatus, system or method of any prior embodiments,wherein the sample comprises reagents configured to detect nucleic acidsby amplifying (generating numerous copies of) the target molecules insamples, wherein target molecule refers to a sequence, or partialsequence, of nucleic acid of interest.

FF3.3. The device, apparatus, system or method of any prior embodiments,wherein the nucleic acid amplification refers to nucleic acidamplification techniques include but not limited to, differentpolymerase chain reaction (PCR) methods, such as hot-start PCR, nestedPCR, touchdown PCR, reverse transcription PCR, RACE PCR, digital PCR,etc., and isothermal amplification methods, such as Loop-mediatedisothermal amplification (LAMP), strand displacement amplification,helicase-dependent amplification, nicking enzyme amplification, rollingcircle amplification, recombinase polymerase amplification, etc.

FF3.4. The device, apparatus, system or method of any prior embodiments,wherein the reagents comprise primers, deoxynucleotides (dNTPs),bivalent cations (e.g. Mg2+), monovalent cation (e.g. K+), buffersolutions, enzymes, or reporters, or any combination or mixture thereof.

FF3.5. The device, apparatus, system or method of any prior embodiments,wherein the reagents are either in the dry form on the inner surface ofthe first or the second plate or both, or in a liquid form encased in,embedded in, or surrounded by, a material that melts with increasingtemperatures, such as, for example, paraffin.

FF3.6. The device, apparatus, system or method of any prior embodiments,wherein primers comprise one or more pairs of forward and reverseprimers.

FF3.7. The device, apparatus, system or method of any prior embodiments,wherein the reagents comprise DNA-dependent polymerase, or RNA-dependentDNA polymerase, or DNA-dependent RNA polymerase.

FF3.8. The device, apparatus, system or method of any prior embodiments,wherein the reagents comprise “reporters” that refer to any tag, label,or dye that can bind to, or intercalate within, the nucleic acidmolecule or be activated by byproducts of the amplification process toenable visualization of the nucleic acid molecule or the amplificationprocess.

FF3.8.1 The device, apparatus, system or method of any priorembodiments, wherein the reports include but are not limited tofluorescent labels or tags or dyes, intercalating agents, molecularbeacon labels, or bioluminescent molecules, or a combination thereof.

FF3.9. The device, apparatus, system or method of any prior embodiments,wherein the reagents comprise cell lysing reagent, which is configuredto facilitate breaking down cellular structures.

FF3.9.1. The device, apparatus, system or method of any priorembodiments, wherein the cell lysing reagent includes but not limited tosalts, detergents, enzymes, and other additives.

FF3.9.2. The device, apparatus, system or method of any priorembodiments, wherein the salt includes but not limited to lithium salt(e.g. lithium chloride), sodium salt (e.g. sodium chloride), potassium(e.g. potassium chloride).

FF3.9.2. The device, apparatus, system or method of any priorembodiments, wherein the detergents are ionic, including anionic andcationic, non-ionic or zwitterionic.

FF3.9.3. The device, apparatus, system or method of any priorembodiments, wherein the ionic detergent includes any detergent which ispartly or wholly in ionic form when dissolved in water.

FF3.9.4. The device, apparatus, system or method of any priorembodiments, wherein anionic detergents include but not limited tosodium dodecyl sulphate (SDS) or other alkali metal alkylsulphate saltsor similar detergents, sarkosyl, or combinations thereof.

FF3.10. The device, apparatus, system or method of any priorembodiments, wherein enzymes includes but not limited to lysozyme,cellulose, and proteinase.

FF3.11. The device, apparatus, system or method of any priorembodiments, wherein chelating agents include but not limited to EDTA,EGTA and other polyamino carboxylic acids, and some reducing agents,such as dithiotreitol (dTT).

FF4. The device, apparatus, system or method of any prior embodiments,wherein the sample comprises an analyte the amount of which is changedwith the temperature changes.

FF5. The device, apparatus, system or method of any prior embodiments,wherein the sample comprises human bodily fluids, such as but notlimited to whole blood, plasma, serum, urine, saliva, and sweat, andcell cultures (mammalian, plant, bacteria, fungi), and a combination ormixture thereof.

FF6. The device, apparatus, system or method of any prior embodiments,wherein the sample is freshly obtained, stored or treated in any desiredor convenient way, for example by dilution or adding buffers, or othersolutions or solvents.

FF7. The device, apparatus, system or method of any prior embodiments,wherein the sample comprises cellular structures such as but not limitedto human cells, animal cells, plant cells, bacteria cells, fungus cells,and virus particles, and a combination or mixture thereof.

GG1. The device, apparatus, system or method of any prior embodiments,wherein an analyte in the sample is stained.

GG2. The device, apparatus, system or method of any prior GGembodiments, wherein the amount of the analyte is measured byfluorescence intensity.

GG3. The device, apparatus, system or method of any prior GGembodiments, wherein the amount of the analyte is measured bycolorimetric intensity.

GG4. The device, apparatus, system or method of any prior embodiments,wherein the analyte is nucleic acid, which is stained with ethidiumbromide (EB), methylene blue, SYBR green I, SYBR green II, pyronin Y,DAPI, acridine orange, or Nancy-520, or a combination thereof.

GG5. The device, apparatus, system or method of any prior embodiments,wherein the analyte is DNA, which is stained with ethidium bromide (EB),methylene blue, pyronin Y, DAPI, acridine orange, or Nancy-520, or acombination thereof, and measured with fluorescence intensity.

GG6. The device, apparatus, system or method of any prior embodiments,wherein the analyte is DNA, which is stained with ethidium bromide (EB),methylene blue, pyronin Y, DAPI, acridine orange, or Nancy-520, or acombination thereof, and measured with colorimetric intensity.

GG7. The device, apparatus, system or method of any prior embodiments,wherein the analyte is RNA, which is stained with ethidium bromide (EB),methylene blue, SYBR green II, pyronin Y, or acridine orange, or acombination thereof, and measured with fluorescence intensity.

GG8. The device, apparatus, system or method of any prior embodiments,wherein the analyte is RNA, which is stained with ethidium bromide (EB),methylene blue, SYBR green II, pyronin Y, or acridine orange, or acombination thereof, and measured with colorimetric intensity.

GG9. The device, apparatus, system or method of any prior embodiments,wherein the analyte is nucleic acid to be detected by reporters.

GG9.1. The device, apparatus, system or method of any prior embodiments,wherein the reporters include but not limited to tag, label, or dye thatcan bind to, or intercalate within, the nucleic acid molecule or beactivated by byproducts of the amplification process to enablevisualization of the nucleic acid molecule or the amplification process.

GG9.2. The device, apparatus, system or method of any prior embodiments,wherein the reporters include but are not limited to fluorescent labelsor tags or dyes, intercalating agents, molecular beacon labels, orbioluminescent molecules, or a combination thereof.

GG9.3. The device, apparatus, system or method of any prior embodiments,wherein the amount of reporter is measured by colorimetric intensityand/or by fluorescence intensity.

HH1. The device, apparatus, system or method of any prior embodiments,wherein the device, apparatus, system or method is configured tofacilitate PCR assays for changing temperature of the sample accordingto a predetermined program.

HH2. The device, apparatus, system or method of any prior embodiments,wherein the device, apparatus, system or method is configured to conductdiagnostic testing, health monitoring, environmental testing, and/orforensic testing.

HH3. The device, apparatus, system or method of any prior embodiments,wherein the device, apparatus, system or method is configured to conductDNA amplification, DNA quantification, selective DNA isolation, geneticanalysis, tissue typing, oncogene identification, infectious diseasetesting, genetic fingerprinting, and/or paternity testing.

HH4. The device, apparatus, system or method of any prior embodiments,wherein the device, apparatus, system or method is configured to conductreal time PCR.

HH5. The device, apparatus, system or method of any prior embodiments,wherein the device, apparatus, system or method is configured to conductnucleic acid amplification.

HH5.1 The device, apparatus, system or method of any prior embodiments,wherein nucleic acid amplification includes any techniques used todetect nucleic acids by amplifying (generating numerous copies of) thetarget molecules in samples, wherein target molecule refers to asequence, or partial sequence, of nucleic acid of interest.

HH6 The device, apparatus, system or method of any prior embodiments,wherein the device, apparatus, system or method is configured to conductnucleic acid amplification techniques include but not limited to,different polymerase chain reaction (PCR) methods, such as hot-startPCR, nested PCR, touchdown PCR, reverse transcription PCR, RACE PCR,digital PCR, etc., and isothermal amplification methods, such asLoop-mediated isothermal amplification (LAMP), strand displacementamplification, helicase-dependent amplification, nicking enzymeamplification, rolling circle amplification, recombinase polymeraseamplification, etc.

A1. A device for rapidly changing temperature of a thin fluidic samplelayer, comprising:

a first plate, a second plate, and a heating/cooling layer, wherein:

the heating/cooling layer is on one of the plates,

each of the plates comprises, on its respective surface, a samplecontact area for contacting a fluidic sample; and

the plates have a configuration for rapidly changing temperature of thesample, in which:

the sample contact areas face each other and are significant parallel,

the average spacing between the contact areas is equal to or less than200 microns,

the two plates regulate (or confine) at least part of the sample into alayer of highly uniform thickness and substantially stagnant relative tothe plates,

the heating/cooling layer is near the at least part of the sample ofuniform thickness,

the area of the at least part of the sample and the heating/coolinglayer are substantially larger than the uniform thickness.

A2. The device of embodiment A1, wherein the heating/cooling layercomprises a disk-coupled dots-on-pillar antenna (D2PA) array, siliconsandwich, graphene, back materials, superlattice or other plasmonicmaterials, other a combination thereof.

A3. The device of embodiment A1, wherein the heating/cooling layercomprises carbon or black nanostructures or a combination thereof.

A4. The device of any of embodiments A1-A3, wherein the heating/coolinglayer is configured to absorb radiation energy.

A5. The device of any of embodiments A1-A4, wherein the heating/coolinglayer is configured to radiate energy in the form of heat afterabsorbing radiation energy.

A6. The device of any of embodiments A1-A5, wherein the heating/coolinglayer is positioned underneath the sample layer and in direct contactwith the sample layer.

A7. The device of any of embodiments A1-A6, wherein the heating/coolinglayer is configured to absorbing electromagnetic waves selected from thegroup consisting of: radio waves, microwaves, infrared waves, visiblelight, ultraviolet waves, X-rays, gamma rays, and thermal radiation.

A8. The device of any of embodiments A1-A7, wherein at least one of theplates does not block the radiation that the heating/cooling layerabsorbs.

A9. The device of any of embodiments A1-A8, wherein one or both of theplates have low thermal conductivity.

A10. The device of any of embodiments A1-A9, wherein the uniformthickness of the sample layer is regulated by one or more spacers thatare fixed to one or both of the plates.

A11. The device of any of embodiments A1-A10, wherein the sample is apre-mixed polymerase chain reaction (PCR) medium.

A12. The device of embodiment A11, wherein the device is configured tofacilitate PCR assays for changing temperature of the sample accordingto a predetermined program.

A13. The device of any of embodiments A1-A12, wherein the device isconfigured to conduct diagnostic testing, health monitoring,environmental testing, and/or forensic testing.

A14. The device of any of embodiments A1-A13, wherein the device isconfigured to conduct DNA amplification, DNA quantification, selectiveDNA isolation, genetic analysis, tissue typing, oncogene identification,infectious disease testing, genetic fingerprinting, and/or paternitytesting.

A15. The device of any of embodiment A1-A14, wherein the sample layer islaterally sealed to reduce sample evaporation.

B1. A system for rapidly changing temperature of a thin fluidic samplelayer, comprising:

a first plate, a second plate, a heating/cooling layer, and a heatingsource, wherein:

the heating/cooling layer is on one of the plates;

the heating source is configured to radiate electromagnetic waves thatthe heating/cooling layer absorbs significantly;

each of the plates comprises, on its respective surface, a samplecontact area for contacting a fluidic sample; and

the plates have a configuration for rapidly changing temperature of thesample, in which:

the sample contact areas face each other and are significant parallel,

the average spacing between the contact areas is equal to or less than200 μm,

the two plates confine at least part of the sample into a layer ofhighly uniform thickness and substantially stagnant relative to theplates,

the heating/cooling layer is near the at least part of the sample ofuniform thickness,

the area of the at least part of the sample and the heating/coolinglayer are substantially larger than the uniform thickness.

B2. The system of embodiment B1, wherein the heating/cooling layercomprises a disk-coupled dots-on-pillar antenna (D2PA) array, siliconsandwich, graphene, superlattice or other plasmonic materials, other acombination thereof.

B3. The system of embodiment B1, wherein the heating/cooling layercomprises carbon or black nanostructures or a combination thereof.

B4. The system of any of embodiments B1-B3, wherein the heating/coolinglayer is configured to absorb at least 80% of the radiation energy fromthe electromagnetic waves from the heating source.

B5. The system of any of embodiments B1-B4, wherein the heating/coolinglayer is configured to radiate energy in the form of heat afterabsorbing radiation energy.

B6. The system of any of embodiments B1-B5, wherein the heating/coolinglayer is positioned underneath the sample layer and in direct contactwith the sample layer.

B7. The system of any of embodiments B1-B6, wherein the heating/coolinglayer is configured to absorbing electromagnetic waves selected from thegroup consisting of: radio waves, microwaves, infrared waves, visiblelight, ultraviolet waves, X-rays, gamma rays, and thermal radiation.

B8. The system of any of embodiments B1-B7, wherein at least one of theplates does not block the radiation from the heating source.

B9. The system of any of embodiments B1-B8, wherein one or both of theplates have low thermal conductivity.

B10. The system of any of embodiments B1-B9, wherein the uniformthickness of the sample layer is regulated by one or more spacers thatare fixed to one or both of the plates.

B11. The system of any of embodiments B1-B10, wherein the sample is apre-mixed polymerase chain reaction (PCR) medium.

B12. The system of embodiment B11, wherein the system is configured tofacilitate PCR assays for changing temperature of the sample accordingto a predetermined program.

B13. The system of any of embodiments B1-B12, wherein the system isconfigured to conduct diagnostic testing, health monitoring,environmental testing, and/or forensic testing.

B14. The system of any of embodiments B1-B15, wherein the system isconfigured to conduct DNB amplification, DNB quantification, selectiveDNB isolation, genetic analysis, tissue typing, oncogene identification,infectious disease testing, genetic fingerprinting, and/or paternitytesting.

B15. The system of any of embodiments B1-B14, wherein the sample layeris laterally sealed to reduce sample evaporation.

B16. The system of any of embodiments B1-B15, further comprising acontroller, which is configured to control the presence, intensity,wavelength, frequency, and/or angle of the electromagnetic waves.

B17. The system of any of embodiments B1-B16, further comprising athermometer, which is configured to measure the temperature at or inproximity of the sample contact area and send a signal to the controllerbased on the measured temperature.

B18. The system of embodiment B17, wherein the thermometer is selectedfrom the group consisting of: fiber optical thermometer, infraredthermometer, liquid crystal thermometer, pyrometer, quartz thermometer,silicon bandgap temperature sensor, temperature strip, thermistor, andthermocouple.

C1. A system for facilitating a polymerase chain reaction (PCR) byrapidly changing temperature of a thin fluidic PCR sample layer,comprising:

a first plate, a second plate, a heating/cooling layer, a heatingsource, and a controller wherein:

the heating/cooling layer is on one of the plates;

the heating source is configured to radiate electromagnetic waves thatthe heating/cooling layer absorbs significantly;

each of the plates comprises, on its respective surface, a samplecontact area for contacting a fluid PCR sample, which is a pre-mixed PCRmedium;

the controller is configured to control the heating source and rapidlychange the temperature of the sample according to a predeterminedprogram; and

the plates have a configuration for rapidly changing temperature of thesample, in which:

the sample contact areas face each other and are significant parallel,

the average spacing between the contact areas is equal to or less than200 μm,

the two plates confine at least part of the sample into a layer ofhighly uniform thickness and substantially stagnant relative to theplates,

the heating/cooling layer is near the at least part of the sample ofuniform thickness, and

the area of the at least part of the sample and the heating/coolinglayer are substantially larger than the uniform thickness.

C2. The system of embodiment C1, wherein the controller is configured tocontrol the present, intensity, wavelength, frequency, and/or angle ofthe electromagnetic waves from the heating source.

C3. The system of embodiment C1 or C2, wherein the heating source andthe heating/cooling layer are configured that the electromagnetic wavescause an average ascending temperature rate ramp of at least 10° C./s;and the removal of the electromagnetic waves results in an averagedescending temperature rate ramp of at least 5° C./s.

C4. The system of any of embodiments C1-C2, wherein the heating sourceand the heating/cooling layer are configured to create an averageascending temperature rate ramp of at least 10° C./s and an averagedescending temperature rate ramp of at least 5° C./s.

C5. The system of any of embodiments C1-C2, wherein the heating sourceand the heating/cooling layer are configured to create an averageascending temperature rate ramp of at least 10° C./s to reach theinitialization step, the denaturation step and/or theextension/elongation step during a PCR, and an average descendingtemperature rate ramp of at least 5° C./s to reach the annealing stepand/or the final cooling step during a PCR.

C6. The system of any of embodiments C1-C5, wherein the PCR samplecomprises: template DNA, primer DNA, cations, polymerase, and buffer.

D1. A method for rapidly changing temperature of a thin fluidic samplelayer, comprising:

providing a first plate a second plate, each of the plates comprising,on its respective inner surface, a sample contact area;

providing a heating/cooling layer and a heating source, wherein theheating/cooling layer is on one of the plates, and the heating source isconfigured to radiate electromagnetic waves that the heating/coolinglayer absorbs significantly;

depositing a fluidic sample on one or both of the plates;

pressing the plates into a closed configuration, in which:

the sample contact areas face each other and are significant parallel,

the average spacing between the contact areas is equal to or less than200 μm,

the two plates confine at least part of the sample into a layer ofhighly uniform thickness and substantially stagnant relative to theplates;

the heating/cooling layer is near the at least part of the sample ofuniform thickness,

the area of the at least part of the sample and the heating/coolinglayer are substantially larger than the uniform thickness; and

changing and maintaining the temperature of the sample layer by changingthe presence, intensity, wavelength, frequency, and/or angle of theelectromagnetic waves from the heating source.

D2. The method of embodiment D1, wherein the step of pressing the platesinto a closed figuration comprises pressing the plates with an imprecisepressing force.

D3. The method of embodiment D1 or D2, wherein the step of pressing theplates into a closed figuration comprises pressing the plates directedlywith human hands.

D4. The method of any of embodiments D1-D3, wherein the layer of highlyuniform thickness has a thickness variation of less than 10%.

D5. The method of any of embodiments D1-D4, wherein the heating/coolinglayer comprises a disk-coupled dots-on-pillar antenna (D2PA) array,silicon sandwich, graphene, superlattice or other plasmonic materials,other a combination thereof.

D6. The method of any of embodiments D1-D5, wherein the heating/coolinglayer comprises carbon or black nanostructures or a combination thereof.

D7. The method of any of embodiments D1-D6, wherein the heating/coolinglayer is configured to absorb at least 80% of the radiation energy fromthe electromagnetic waves from the heating source.

D8. The method of any of embodiments D1-D7, wherein the heating/coolinglayer is configured to radiate energy in the form of heat afterabsorbing radiation energy.

D9. The method of any of embodiments D1-D8, wherein the heating/coolinglayer is positioned underneath the sample layer and in direct contactwith the sample layer.

D10. The method of any of embodiments D1-D9, wherein the heating/coolinglayer is configured to absorbing electromagnetic waves selected from thegroup consisting of: radio waves, microwaves, infrared waves, visiblelight, ultraviolet waves, X-rays, gamma rays, and thermal radiation.

D11. The method of any of embodiments D1-D10, wherein at least one ofthe plates does not block the radiation from the heating source.

D12. The method of any of embodiments D1-D11, wherein one or both of theplates have low thermal conductivity.

D13. The method of any of embodiments D1-D12, wherein the uniformthickness of the sample layer is regulated by one or more spacers thatare fixed to one or both of the plates.

D14. The method of any of embodiments D1-D13, wherein the sample is apre-mixed polymerase chain reaction (PCR) medium.

D15. The method of embodiment D14, wherein the method is used tofacilitate PCR assays for changing temperature of the sample accordingto a predetermined program.

D16. The method of any of embodiments D1-D15, wherein the method is usedto conduct diagnostic testing, health monitoring, environmental testing,and/or forensic testing.

D17. The method of any of embodiments D1-D16, wherein the method is usedto conduct DNB amplification, DNB quantification, selective DNBisolation, genetic analysis, tissue typing, oncogene identification,infectious disease testing, genetic fingerprinting, and/or paternitytesting.

D18. The method of any of embodiments D1-D17, wherein the sample layeris laterally sealed to reduce sample evaporation.

D19. The method of any of embodiments D1-D18, wherein the heating sourceis controlled by a controller, which is configured to control thepresence, intensity, wavelength, frequency, and/or angle of theelectromagnetic waves.

D20. The method of any of embodiments D1-D19, wherein the controller isconfigured to receive signals from a thermometer, which is configured tomeasure the temperature at or in proximity of the sample contact areaand send a signal to the controller based on the measured temperature.

D21. The method of embodiment D20, wherein the thermometer is selectedfrom the group consisting of: fiber optical thermometer, infraredthermometer, liquid crystal thermometer, pyrometer, quartz thermometer,silicon bandgap temperature sensor, temperature strip, thermistor, andthermocouple.

E1. A method for facilitating a polymerase chain reaction (PCR) byrapidly changing temperatures in a fluidic PCR sample, comprising:

providing a first plate a second plate, each of the plates comprising,on its respective inner surface, a sample contact area;

providing a heating/cooling layer, a heating source and a controller,wherein the heating/cooling layer is on one of the plates, and theheating source is configured to radiate electromagnetic waves that theheating/cooling layer absorbs significantly;

depositing a fluidic PCR sample on one or both of the plates;

pressing the plates into a closed configuration, in which:

the sample contact areas face each other and are significant parallel,

the average spacing between the contact areas is equal to or less than200 μm,

the two plates confine at least part of the PCR sample into a layer ofhighly uniform thickness and substantially stagnant relative to theplates;

the heating/cooling layer is near the at least part of the PCR sample ofuniform thickness,

the area of the at least part of the sample and the heating/coolinglayer are substantially larger than the uniform thickness; and

using the controller to control the heating source to conduct a PCR bychanging and maintaining the temperature of the PCR sample layeraccording to a predetermined program, wherein when the temperatures arechanged, the heating source creates an average ascending temperaturerate ramp of at least 10° C./s and an average descending temperaturerate ramp of at least 5° C./s during the PCR.

E2. The method of embodiment E1, wherein changing and maintaining thetemperature of the PCR sample layer is achieved by adjusting theintensity, wavelength, frequency, and/or angle of the electromagneticwaves from the heating source.

E3. The system of any of embodiments E1-E2, wherein the heating sourceand the heating/cooling layer are configured to create an averageascending temperature rate ramp of at least 10° C./s to reach theinitialization step, the denaturation step and/or theextension/elongation step during a PCR, and an average descendingtemperature rate ramp of at least 5° C./s to reach the annealing stepand/or the final cooling step during a PCR.

E4. The method of any of embodiments E1-E3, wherein the PCR samplecomprises: template DNA, primer DNA, cations, polymerase, and buffer.

NN1 A device for rapidly changing temperature of a thin fluidic samplelayer, comprising:

a first plate, and a second plate, wherein:

each of the plates comprises, on its respective surface, a samplecontact area for contacting a fluidic sample; and

the plates have a configuration for rapidly changing temperature of thesample, in which:

the sample contact areas face each other and are significant parallel,

the average spacing between the contact areas is equal to or less than200 microns,

the two plates regulate (or confine) at least part of the sample into alayer of highly uniform thickness and substantially stagnant relative tothe plates,

the heating/cooling layer is near the at least part of the sample ofuniform thickness,

the area of the at least part of the sample and the heating/coolinglayer are substantially larger than the uniform thickness.

JJ1. The device of any prior embodiments, further comprising a hingethat connects the first plate and the second plate, and is configured toallow the two plates to rotate around the hinge into differentconfigurations.

JJ2. The device of any prior embodiments, wherein after relativeposition of the plates are adjusted by an external force, the hingemaintains an angle between the two plates that is within 5 degrees fromthe angle just before the external force is removed.

JJ3. The device of any prior embodiments, wherein the wherein afterrelative position of the plates are adjusted by an external force, thehinge maintains an angle between the two plates that is within 10degrees from the angle just before the external force is removed.

JJ4. The device of any prior embodiments, wherein the hinge is made of apiece of a piece of hinge material of a substantially uniform thickness,wherein the hinge material is attached to a part of the inner surface ofthe first plate and a part of the outer surface of the second plate, andthe attachments do not completely separate using operation.

JJ5. The device of any prior embodiments, wherein the hinge is made of apiece of hinge material of a substantially uniform thickness, whereinthe hinge material is attached a part of the outer surfaces of the firstplate and the second plate, and the attachments do not completelyseparate using operation.

JJ6. The device of any prior embodiments, wherein the hinge material isa metal.

JJ7. The device of any prior embodiments, wherein the hinge materials isselected from a group consisting of: gold, silver, copper, aluminum,iron, tin, platinum, nickel, cobalt, and alloys thereof.

JJ8. The device of any prior embodiments, wherein the hinge comprises afirst leaf, a second leaf, and a joint that connects the leaves and isconfigured for the leaves to rotate around the joint,

wherein the first leaf is attached to the first plate inner surfacewithout wrapping around any edge of the first plate, the second leaf isattached to the second plate outer surface, and the joint is positionedlongitudinally parallel to the hinge edge of the second plate, allowingthe two plates to rotate around the joint

KK1. The device of any prior embodiments, wherein:

one of the plate comprises one or more open notches on an edge orcorners of the plate, and

at or near the closed configuration, an edge of the other plate isconfigured to overlap with the open notch

KK2. The device of any prior embodiments, wherein the notch facilitateschanging the plates from a configuration that is near or at closedconfiguration to an open configuration for sample deposition.

KK3. The device of any prior embodiments, wherein the width of at leastone notch is in the range of ⅙ to ⅔ of the width of the notched edge.

KK4. The device of any prior embodiments, wherein the opening edge ofthe plate without the notch is inside the notched edge except for thepart over the notch.

KK5. The device of any prior embodiments, wherein the first platecomprises one or more notched edges, each of which has at least onenotch; and the second plate comprises one or more corresponding openingedges juxtaposed over the notches, allowing a user to push against oneof the opening edges over the notch to switch the two plates between theclosed configuration and the open configuration or to change the angleformed by the first plate and the second plate

KK6. The device of any prior embodiments, wherein the notch ispositioned at an intersection of two neighboring notched edges.

LL1. The device of any prior embodiments, wherein any prior deviceembodiment, wherein each of the plate further comprises, on itsrespective outer surface, a force area for applying a pressing forcethat forces the plates together, and wherein the force is an impreciseforce that has a magnitude which is, at the time that the force isapplied, either (a) unknown and unpredictable, or (b) cannot be knownand cannot be predicted within an accuracy equal or better than 30% ofthe force applied.

LL2. The device of any prior embodiments, wherein each of the platefurther comprises, on its respective outer surface, a force area forapplying a pressing force that forces the plates together, and whereinthe force is an imprecise force that has a magnitude which cannot, atthe time that the force is applied, be determined within an accuracyequal or better than 30%, 40%, 50%, 70%, 100%, 200%, 300%, 500%, 1000%,2000%, or any range between the two values.

LL3. The device of any prior embodiments, wherein the imprecise force isprovided by human hand.

MM1. The device, apparatus, system, or method of any prior embodiments,wherein the first plate and the second plate are flexible plastic filmand/or thin glass film, that each has a substantially uniform thicknessof a value selected from a range between 1 um to 25 um.

MM2. The device, apparatus, system, or method of any prior embodiments,wherein each plate has an area in a range of 1 cm{circumflex over ( )}2to 16 cm{circumflex over ( )}2.

MM3. The device, apparatus, system, or method of any prior embodiments,wherein the sample sandwiched between the two plate has a thickness of40 um or less.

MM4. The device, apparatus, system, or method of any prior embodiments,wherein the relevant sample to the entire sample ratio (RE ratio) is 12%or less.

MM5. The device, apparatus, system, or method of any prior embodiments,wherein the cooling zone is at least 9 times larger than the heatingzone.

MM6. The device, apparatus, system, or method of any prior embodiments,wherein the sample to non-sample thermal mass ratio is 2.2 or lager.

MM7. The device, apparatus, system, or method of any prior embodiments,wherein the RHC card does not comprise spacer.

MM8. The device, apparatus, system, or method of any prior embodiments,wherein the RHC card comprises spacers that are fixed on one or both ofthe plates.

MM9. The device, apparatus, system, or method of any prior embodiments,wherein:

the first plate and second plates are plastic or a thin glass. The firstplate and second plate have a thickness of 100 nm, 500 nm, 1 um, 5 um,10 um, in a range between any of the two values;

the sample between the two plates has a thickness of 5 um, 10 um, 30 um,50 um, 100 um, or in a range between any of the two values;

the distance from the H/C layer to the sample is 10 nm, 100 nm, 500 nm,1 um, 5 um, 10 um, or in a range between any of the two values;

the ratio of the cooling zone area to the relevant sample area is 16, 9,4, 2, or in a range between any of the two values;

the ratio of the cooling zone area to the heating area is 16, 9, 4, 2,or in a range between any of the two values; AND

the distance between the H/C layer and the heating source (e.g. LED) is5 mm, 10 mm, 20 mm, 30 mm, or in a range between any of the two values.

MM10. The device, apparatus, system, or method of any prior embodiments,wherein:

the first plate and second plates are plastic or a thin glass. The firstplate and second plate have a thickness of 100 nm, 500 nm, 1 um, 5 um,10 um, in a range between any of the two values;

the sample between the two plates has a thickness of 5 um, 10 um, 30 um,50 um, 100 um, or in a range between any of the two values;

the distance from the H/C layer to the sample is 10 nm, 100 nm, 500 nm,1 um, 5 um, 10 um, or in a range between any of the two values;

the ratio of the cooling zone area to the relevant sample area is 16, 9,4, 2, or in a range between any of the two values;

the ratio of the cooling zone area to the heating area is 16, 9, 4, 2,or in a range between any of the two values; OR

the distance between the H/C layer and the heating source (e.g. LED) is5 mm, 10 mm, 20 mm, 30 mm, or in a range between any of the two values.

MM11. The device, apparatus, system, or method of any prior embodiments,wherein:

the first plate and second plates are plastic or a thin glass. The firstplate has a thickness of 10 um, 25 um, 50 um, or in a range between anyof the two values; while the second plate (that plate that has heatinglayer or cooling layer) has a thickness of 100 nm, 500 nm, 1 um, 5 um,10 um, in a range between any of the two values;

the sample between the two plates has a thickness of 5 um, 10 um, 30 um,50 um, 100 um, or in a range between any of the two values;

the distance between the H/C layer and the sample is 10 nm, 100 nm, 500nm, 1 um, 5 um, 10 um, or in a range between any of the two values;

the ratio of the cooling zone area to the relevant sample area is 16, 9,4, 2, or in a range between any of the two values;

the ratio of the cooling zone area to the heating area is 16, 9, 4, 2,or in a range between any of the two values; AND

the distance between the H/C layer and the heating source (e.g. LED) is5 mm, 10 mm, 20 mm, 30 mm, or in a range between any of the two values.

MM12. The device, apparatus, system, or method of any prior embodiments,wherein:

the first plate and second plates are plastic or a thin glass. The firstplate has a thickness of 10 um, 25 um, 50 um, or in a range between anyof the two values; while the second plate (that plate that has heatinglayer or cooling layer) has a thickness of 100 nm, 500 nm, 1 um, 5 um,10 um, in a range between any of the two values;

the sample between the two plates has a thickness of 5 um, 10 um, 30 um,50 um, 100 um, or in a range between any of the two values;

the distance between the H/C layer and the sample is 10 nm, 100 nm, 500nm, 1 um, 5 um, 10 um, or in a range between any of the two values;

the ratio of the cooling zone area to the relevant sample area is 16, 9,4, 2, or in a range between any of the two values;

the ratio of the cooling zone area to the heating area is 16, 9, 4, 2,or in a range between any of the two values; OR

the distance between the H/C layer and the heating source (e.g. LED) is5 mm, 10 mm, 20 mm, 30 mm, or in a range between any of the two values.

MM13. The device, apparatus, system, or method of any prior embodiments,wherein:

the first plate and second plates are plastic or a thin glass. The firstplate and second plate have a thickness of 100 nm, 500 nm, 1 um, 5 um,10 um, 25 um, 50 um, 100 um, 175 um, 250 um, or in a range between anyof the two values;

the sample between the two plates has a thickness of 100 nm, 500 nm, 1um, 5 um, 10 um, 25 um, 50 um, 100 um, 250 um, or in a range between anyof the two values;

the distance between the H/C layer and the sample is 100 nm, 500 nm, 1um, 5 um, 10 um, 25 um, 50 um, 100 um, 175 um, 250 um, or in a rangebetween any of the two values;

the ratio of the cooling zone area to the relevant sample area is 100,64, 16, 9, 4, 2, 1, 0.5, 0.1, or in a range between any of the twovalues;

the ratio of the cooling zone area to the heating zone is 100, 64, 16,9, 4, 2, 1, 0.5, 0.1, or in a range between any of the two values; AND

the distance between the H/C layer and the heating source (e.g. LED) is500 um, 1 mm, 3 mm, 5 mm, 10 mm, 20 mm, 30 mm, or in a range between anyof the two values.

MM14. The device, apparatus, system, or method of any prior embodiments,wherein:

the first plate and second plates are plastic or a thin glass. The firstplate and second plate have a thickness of 100 nm, 500 nm, 1 um, 5 um,10 um, 25 um, 50 um, 100 um, 175 um, 250 um, or in a range between anyof the two values;

the sample between the two plates has a thickness of 100 nm, 500 nm, 1um, 5 um, 10 um, 25 um, 50 um, 100 um, 250 um, or in a range between anyof the two values;

the distance between the H/C layer and the sample is 100 nm, 500 nm, 1um, 5 um, 10 um, 25 um, 50 um, 100 um, 175 um, 250 um, or in a rangebetween any of the two values;

the ratio of the cooling zone area to the relevant sample area is 100,64, 16, 9, 4, 2, 1, 0.5, 0.1, or in a range between any of the twovalues;

the ratio of the cooling zone area to the heating zone is 100, 64, 16,9, 4, 2, 1, 0.5, 0.1, or in a range between any of the two values; OR

the distance between the H/C layer and the heating source (e.g. LED) is500 um, 1 mm, 3 mm, 5 mm, 10 mm, 20 mm, 30 mm, or in a range between anyof the two values.

MM15. The device, apparatus, system, or method of any prior embodiments,wherein a light pipe collimates the light from a light source (e.g. LED)into the heating zone; the light pipe comprises a structure with ahollow hole (e.g. a tube or a structure milled a hole) with a reflectivewall; and the light pipe has a lateral dimension for 1 mm to 8 mm andlength of 2 mm to 5o mm.

MM16. The device, apparatus, system, or method of any prior embodiments,wherein:

the first plate and second plates are plastic or a thin glass;

the first plate and second plate have a thickness of 100 nm, 500 nm, 1um, 5 um, 10 um, in a range between any of the two values;

the sample between the two plates has a thickness in a range of 1 to 5um, 5 um to 10 um, 10 to 30 um, or 30 um to 50 um;

the distance from the H/C layer to the sample is in a range of 10 nm to100 nm, 100 nm to 500 nm, 500 nm to 1 um, 1 um to 5 um, 5 um to 10 um,or 10 um to 25 um;

the ratio of the cooling zone area to the relevant sample area is 16, 9,4, 2, or in a range between any of the two values;

the ratio of the cooling zone area to the heating area is 16, 9, 4, 2,or in a range between any of the two values;

the distance between the H/C layer and the heating source (e.g. LED) is5 mm, 10 mm, 20 mm, 30 mm, or in a range between any of the two values;

the KC ratio for the cooling layer is in a range of between 0.5 cm²/secand 0.7 cm²/sec, 0.7 cm²/sec and 0.9 cm²/sec, 0.9 cm²/sec and 1 cm²/sec,1 cm²/sec and 1.1 cm²/sec, 1.1 cm²/sec and 1.3 cm²/sec, 1.3 cm²/sec and1.6 cm²/sec, 1.6 cm²/sec and cm²/sec, or 2 cm²/sec and cm²/sec; and

the sample to non-sample thermal mass ratio is in a range of between 0.2to 0.5, 0.5 to 0.7, 0.7 to 1, 1 to 1.5, 1.5 to 5, 5 to 10, 10 to 30, 30to 50, or 50 to 100.

MM17. The device, apparatus, system, or method of any prior embodiments,wherein:

the first plate and second plates are plastic or a thin glass;

the first plate and second plate have a thickness of 100 nm, 500 nm, 1um, 5 um, 10 um, in a range between any of the two values;

the sample between the two plates has a thickness in a range of 1 to 5um, 5 um to 10 um, 10 to 30 um, or 30 um to 50 um;

the distance from the H/C layer to the sample is in a range of 10 nm to100 nm, 100 nm to 500 nm, 500 nm to 1 um, 1 um to 5 um, 5 um to 10 um,or 10 um to 25 um;

the ratio of the cooling zone area to the relevant sample area is 16, 9,4, 2, or in a range between any of the two values;

the ratio of the cooling zone area to the heating area is 16, 9, 4, 2,or in a range between any of the two values;

the distance between the H/C layer and the heating source (e.g. LED) is5 mm, 10 mm, 20 mm, 30 mm, or in a range between any of the two values;

the KC ratio for the cooling layer is in a range of between 0.5 cm²/secand 0.7 cm²/sec, 0.7 cm²/sec and cm²/sec, 0.9 cm²/sec and 1 cm²/sec, 1cm²/sec and 1.1 cm²/sec, 1.1 cm²/sec and 1.3 cm²/sec, 1.3 cm²/sec and1.6 cm²/sec, 1.6 cm²/sec and 2 cm²/sec, or 2 cm²/sec and 3 cm²/sec; OR

the sample to non-sample thermal mass ratio is in a range of between 0.2to 0.5, 0.5 to 0.7, 0.7 to 1, 1 to 1.5, 1.5 to 5, 5 to 10, 10 to 30, 30to 50, or 50 to 100.

NN1. The device, apparatus, system or method of any prior embodiments,wherein the device comprises a heating layer and a cooling layer, wherethe cooling layer has an area larger than that heating zone.

NN2. The device, apparatus, system or method of any prior embodiments,wherein the device comprises one heating/cooling layer, where thecooling zone has an area larger than that heating zone.

NN3. The device, apparatus, system or method of any prior embodiments,wherein the device comprises a cooling layer that has a high thermalconductivity (50 W/(m²·-K)) and an area larger than lateral area of arelevant sample.

NN4. The device, apparatus, system or method of any prior embodiments,wherein the device comprises a cooling layer that has a high thermalconductivity (greater than 50 W/(m²·K)(m·K)) and an area larger thanlateral area of a relevant sample by a factor of 2 to 40.

NN5. The device, apparatus, system or method of any prior embodiments,wherein the device comprises a cooling layer that has (i) a high thermalconductivity (greater than 50 W/(m·K)), and (ii) thermal radiationenhancement layer (specify the thermal radiation).

NN6. The device, apparatus, system or method of any prior embodiments,wherein the device comprises a cooling layer that has (i) a high thermalconductivity (greater than 50 W/(m·K)), and (ii) thermal radiationenhancement layer, and (iii) an area larger than lateral area of arelevant sample.

NN7. The device, apparatus, system or method of any prior embodiments,wherein the device comprises a cooling layer that has (i) a high thermalconductivity (greater than 50 W/(m·K)), and (ii) thermal radiationenhancement layer, and (iii) an area larger than lateral area of arelevant sample by a factor of 1.5 to 100.

NN8. The device, apparatus, system or method of any prior embodiments,wherein the device comprises a cooling zone has a thermal radiationenhancement layer that has an average light absorption coefficient of70% over the wavelength range.

NN9. The device, apparatus, system or method of any prior embodiments,wherein the device comprises a cooling zone has a thermal conductivitymultiplying its thickness in the range of 6×10⁻⁵ W/K to 3×10⁻⁴ W/K.

NN10. The device, apparatus, system or method of any prior embodiments,wherein the device comprises a cooling zone comprises a gold layer of athickness in the range of 200 nm to 800 nm.

NN11. The device, apparatus, system or method of any prior embodiments,wherein the device comprises a thermal conductivity multiplying itsthickness in the range of 6×10⁻⁵ W/K to 3×10⁻⁴ W/K.

NN12. The device, apparatus, system or method of any prior embodiments,wherein the device comprises a cooling layer that:

has a high thermal conductivity (greater than 50 W/(m·K)),

comprises thermal radiation enhancement layer that has an average lightabsorption coefficient of 70% over the wavelength range;

has an area larger than lateral area of a relevant sample by a factor of1.5 to 100; and

has a thermal conductivity multiplying its thickness in the range of6×10-5 W/K to 3×10−4 W/K.

NN13. The device, apparatus, system or method of any prior embodiments,wherein the device comprises a cooling zone (layer) has thermalconductivity times its thickness of 6×10⁻⁵ W/K, 9×10⁻⁵ W/K, 1.2×10⁻⁴W/K, 1.5×10⁻⁴ W/K, 1.8×10⁻⁴ W/K, 2.1×10⁻⁴ W/K, 2.7×10⁻⁴ W/K, 3×10⁻⁴ W/K,1.5×10⁻⁴ W/K, or in a range between any of the two values.

NN14. The device, apparatus, system or method of any prior embodiments,wherein the device comprises a cooling zone (layer) has thermalconductivity times its thickness in a range of 6×10⁻⁵ W/K to 9×10⁻⁵ W/K,9×10⁻⁵ W/K to 1.5×10⁻⁴ W/K, 1.5×10⁻⁴ W/K to 2.1×10⁻⁴ W/K, 2.1×10⁻⁴ W/Kto 2.7×10⁻⁴ W/K, 2.7×10⁻⁴ W/K to 3×10⁻⁴ W/K, or 3×10⁻⁴ W/K to 1.5×10⁻⁴W/K.

NN15. The device, apparatus, system or method of any prior embodiments,wherein the device comprises cooling zone (layer) has thermalconductivity times its thickness in a range of 9×10⁻⁵ W/K to 2.7×10⁻⁴W/K, 9×10⁻⁵ W/K to 2.4×10⁻⁴ W/K, 9×10⁻⁵ W/K to 2.1×10⁻⁴ W/K, or 9×10⁻⁵W/K to 1.8×10⁻⁴ W/K.

NN16. The device, apparatus, system or method of any prior embodiments,wherein the device comprises cooling zone comprises a gold layer of athickness in the range of 200 nm to 800 nm. In another embodiment, acooling zone comprises a gold layer of a thickness in the range of 300nm to 700 nm.

NN17. The device, apparatus, system or method of any prior embodiments,wherein in the device the materials between the heating zone and therelevant sample has a thermal conductivity and a thickness configured tohave a conductance per unit area that is equal to or larger than 1,000W/(m²·K), 2000 W/(m²·K), 3,000 W/(m²·K), 4000 W/(m²·K), 5000 W/(m²·K),7,000 W/(m²·K), 10,000 W/(m²·K), 20,000 W/(m²·K), 50,000 W/(m²·K),50,000 W/(m²·K), 100,000 W/(m²·K), or in a range of any the values.

NN18. The device, apparatus, system or method of any prior embodiments,wherein a preferred conductance per unit area of the material betweenthe heating zone and the relevant sample is in a range of 1,000 W/(m²·K)to 2,000 W/(m²·K), 2,000 W/(m²·K) to 4000 W/(m²·K), 4,000 W/(m²·K) to10,000 W/(m²·K), or 10,000 W/(m²·K) to 100,000 W/(m²·K).

NN19. The device, apparatus, system or method of any prior embodiments,wherein there is zero distance between the heating zone and the relevantsample, and hence an infinity for the conductance per unit area of thematerial between the heating zone and the relevant sample.

NN20. The device, apparatus, system or method of any prior embodiments,wherein the heating layer or the cooling layer is separated from arelevant sample by a thin plastics plate (or film) which has a thermalconductivity in the range of 0.1 to 0.3 W/(m·K), and the thin plasticlayer has a thickness of 0 nm, 10 nm, 50 nm, 100 nm, 200 nm, 300 nm, 400nm, 500 nm, 1 um, 2.5 um, 5 um, 10 um, 25 um, 50 um, 75 nm 100 um, 150um, or in a range between any of the two values

NN21. The device, apparatus, system or method of any prior embodiments,wherein the thin plastic plate (or film) that separate the relevantsample from the heating layer or the cooling layer has thickness in arange between 0 nm and 100 nm, 100 nm and 500 nm, 500 nm and 1 um, 1 umand 5 um, 5 um and 10 um, 10 um and 25 um, 25 um and 50 um, 50 um and 75um, 75 um and 100 um, or 100 um and 150 um.

NN22. The device, apparatus, system or method of any prior embodiments,wherein the thin plastic plate (or film) that separates the relevantsample from the heating layer or the cooling layer has thickness of 0.1um, 0.5 um, 1 um, 5 um, 10 um, 20 um, 25 um, or a range between any twovalues.

NN23. The device, apparatus, system or method of any prior embodiments,wherein the area of the heating zone is only a fraction of the area ofthe cooling zone or area, and the area of the cooling zone (layer) islarger than the area of the heating zone by a factor of 1.1, 1.5, 2, 3,4, 5, 10, 20, 30, 40, 50, 70, 100, 200, 300, 400, 500, 600, 700, 800,800, 1,000, 5000, 10,000, 100,000, or in a range between any of the twovalues.

NN24. The device, apparatus, system or method of any prior embodiments,wherein the cooling zone (layer) has an area that is larger than thelateral area of the hearing zone (layer) by a factor in a range of 1.1to 1.5, 1.5 to 5, 5 to 10, 10 to 50, 50 to 100, 100 to 500, 500 to1,000, 1000, to 10,000, or 10,000 to 100,000.

NN25. The device, apparatus, system or method of any prior embodiments,wherein the cooling zone (layer) has an area that is larger than thelateral area of the relevant sample by a factor of 1.5, 2, 3, 4, 5, 10,20, 50, 70, 100, 200, 300, 400, 500, 600, 700, 800, 800, 1,000, 2000,5000, 10,000, 100,000, or in a range between any of the two values.

NN26. The device, apparatus, system or method of any prior embodiments,wherein the cooling zone (layer) has an area that is larger than thelateral area of the relevant sample by a factor in a range of 1.5 to 5,5 to 10, 10 to 50, 50 to 100, 100 to 500, 500 to 1,000, 1000, to 10,000,or 10,000 to 100,000.

NN27. The device, apparatus, system or method of any prior embodiments,wherein the first plate or the second plate has a thickness of 10 nm,100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 1 um, 2.5 um, 5 um, 10 um, 25um, 50 um, 100 um, 200 um, or 500 um, 1000 um, or in a range between anyof the two values.

NN28. The device, apparatus, system or method of any prior embodiments,wherein the first plate and the second plate can have the same thicknessor a different thickness, and can be made of the same materials ordifferent materials.

NN29. The device, apparatus, system or method of any prior embodiments,wherein the first plate or the second plate has a thickness in a rangeof between 10 nm and 500 nm, 500 nm and 1 um, 1 um and 2.5 um, 2.5 umand 5 um, 5 um and 10 um, 10 um and 25 um, um and 50 um, 50 um and 100um, 100 um and 200 um, or 200 um and 500 um, or 500 um and 1000 um.

NN30. The device, apparatus, system or method of any prior embodiments,wherein the first plate and second plates are plastic, a thin glass, ora material with similar physical properties. The first plate or secondplate have a thickness of 100 nm, 500 nm, 1 um, 5 um, 10 um, 25 um, 50um, 100 um, 175 um, 250 um, or in a range between any of the two values.

NN31. The device, apparatus, system or method of any prior embodiments,wherein the ratio of the average lateral size of the relevant samplevolume to the diffusion length of the reagent during the time forthermal cycling or a reaction is equal to or larger than 5, 6, 7, 10,15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, 1,000,5,000, 10,000, 100,000, or in a range between any two values.

NN32. The device, apparatus, system or method of any prior embodiments,wherein the ratio of the average lateral size of the relevant samplevolume to the diffusion length of the reagent during the time forthermal cycling or a reaction is in a range of 5 to 10, 10 to 30, 30 to60, 6 to 100, 100 to 200, 200 to 500, 500 to 1,000, 1,000 to 5000, 5,000to 10,000, or 10,000 to 100,000.

NN33. The device, apparatus, system or method of any prior embodiments,wherein the ratio of the average lateral size of the relevant samplevolume to the diffusion length of the reagent during the time forthermal cycling or a reaction is in a range of 5 to 10, 10 to 30, 30 to60, 6 to 100, 100 to 200, 200 to 500, 500 to 1,000, 1,000 to 5,000,5,000 to 10,000, or 10,000 to 100,000.

NN34. The device, apparatus, system or method of any prior embodiments,wherein the average lateral dimension of the relevant volume is 1 mm, 2mm, 3 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm 10 mm, 12 mm, 15 mm, 20 mm, 30mm, 40 mm, 50 mm, 70 mm, 100 mm, 200 mm, or in a range between any twovalues.

NN35. The device, apparatus, system or method of any prior embodiments,wherein the average lateral dimension of the relevant volume is in arange of 1 mm to 5 mm, 5 mm to 10 mm, 10 mm to 20 mm, 20 mm to 40 mm, 40mm to 70 mm, 70 mm to 100 mm, or 100 mm to 200 mm.

NN36. The device, apparatus, system or method of any prior embodiments,wherein the average lateral dimension of the relevant volume is in arange of 1 mm to 5 mm, 1 mm to 10 mm, or 5 mm to 20 mm.

NN37. The device, apparatus, system or method of any prior embodiments,wherein the thermal radiation enhancement surface has a high averagelight absorptance (e.g. the black paint used in our experiments). Incertain embodiments, the cooling zone has a surface that has an averagelight absorptance of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, or ina range between any of the two values.

NN38. The device, apparatus, system or method of any prior embodiments,wherein the cooling zone has a surface that has an average lightabsorptance in a range of 30% to 40%, 40% to 60%, 60% to 80% to 90%, or90% to 100%.

NN39. The device, apparatus, system or method of any prior embodiments,wherein the cooling zone has a surface that has an average lightabsorptance in a range of 30% to 100%, 50% to 100%, 70% to 100%, or 80%to 100%.

NN40. The device, apparatus, system or method of any prior embodiments,wherein the cooling zone has a surface that has an average lightabsorptance of a value given above by averaging over a wavelength range400 nm to 800 nm, 700 nm to 1500 nm, 900 nm to 2,000 nm, or 2,000 nm to20,000 nm.

NN41. The device, apparatus, system or method of any prior embodiments,wherein the black paints are polymer mixtures that look black by humaneyes. A black paint include, but not limited to, a mixture of polymersand nanoparticles. One example of the nanoparticles is black carbonnanoparticle, carbon, nanotubes, graphite particles, graphene, metalnanoparticles, semiconductor nanoparticles, or a combination thereof.

NN42. The device, apparatus, system or method of any prior embodiments,wherein the plasmonic structures include nanostructured plasmonicstructures.

NN43. The device, apparatus, system or method of any prior embodiments,wherein a cooling plate comprise a layer of high thermal conductivitymetal (50 W/(m·K) or higher) with a surface thermal radiationenhancement layer. In some embodiments, the surface thermal radiationenhancement layer has a low lateral thermal conductance, which is due toeither ultrathin layer, low thermal conductivity, or both.

NN44. The device, apparatus, system or method of any prior embodiments,wherein thermal radiative cooling is achieved by increasing the area ofradiative cooling layer (i.e. a high-K material, unless statedotherwise), and the radiative cooling layer area is larger than thelateral area of the relevant sample by a factor of 1.2, 1.5, 2, 3, 4, 5,10, 20, 30, 40, 50, 60, 70, 80 100, 200, 300, 400, 500, 600, 700, 800,800, 1,000, 2,000, 5,000, 10,000, 100,000, or in a range between any ofthe two values.

NN45. The device, apparatus, system or method of any prior embodiments,wherein the radiative cooling zone (layer) has an area that is largerthan the lateral area of the relevant sample by a factor in a range of1.2 to 3, 3 to 5, 5 to 10, 10 to 50, 50 to 100, 100 to 500, 500 to1,000, 1000, to 10,000, or 10,000 to 100,000.

NN46. The device, apparatus, system or method of any prior embodiments,wherein the ratio of the thermal radiation cooling by the cooling zone(layer) to the total cooling of the sample and sample holder during athermal cycling is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, orin a range between any of the two values.

NN47. The device, apparatus, system or method of any prior embodiments,wherein the ratio of the thermal radiation cooling by the cooling zone(layer) to the total cooling of the sample and sample holder during athermal cycling is in a range of between 10% and 20%, 20% and 30%, 30%and 40%, 40% and 50%, 50% and 60%, 60% and 70%, 70% and 80%, 80% and90%, or 90% and 99%.

NN48. The device, apparatus, system or method of any prior embodiments,wherein the KC ratio materials for the heating layer is equal to orhigher than 0.1 cm²/sec, 0.2 cm²/sec, 0.3 cm²/sec, 0.4 cm²/sec, 0.5cm²/sec, 0.6 cm²/sec, 0.7 cm²/sec, 0.8 cm²/sec, 0.9 cm²/sec, 1 cm²/sec,1.1 cm²/sec, 1.2 cm²/sec, 1.3 cm²/sec, 1.4 cm²/sec, 1.5 cm²/sec, 1.6cm²/sec, 2 cm²/sec, 3 cm²/sec, or in a range between any of the twovalues.

NN49. The device, apparatus, system or method of any prior embodiments,wherein the KC ratio for the heating layer is in a range of between 0.5cm²/sec and 0. cm²/sec, 0.7 cm²/sec and 0.9 cm²/sec, 0.9 cm²/sec and 1cm²/sec, 1 cm²/sec and 1.1 cm²/sec, 1.1 cm²/sec and 1.3 cm²/sec, 1.3cm²/sec and 1.6 cm²/sec, 1.6 cm²/sec and 2 cm²/sec, or 2 cm²/sec and 3cm²/sec.

NN50. The device, apparatus, system or method of any prior embodiments,wherein a thermal radiation enhancement surface(s) will be used (on oneside or both side of the heating zone). A thermal radiation absorptionenhancement surface can be achieved by directly modify the structures ofthe surface (e.g. patterning nanostructures), coating a high thermalradiation materials (e.g. coating a black paint), or both.

NN51. The device, apparatus, system or method of any prior embodiments,wherein the thermal radiation enhancement surface has a high averagelight absorptance (e.g. the black paint used in our experiments). Incertain embodiments, the heating zone has a surface that has an averagelight absorptance of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, or ina range between any of the two values.

NN52. The device, apparatus, system or method of any prior embodiments,wherein the heating zone has a surface that has an average lightabsorptance in a range of 30% to 40%, 40% to 60%, 60% to 80% to 90%, or90% to 100%.

NN53. The device, apparatus, system or method of any prior embodiments,wherein the heating zone has a surface that has an average lightabsorptance in a range of 30% to 100%, 50% to 100%, 70% to 100%, or 80%to 100%.

NN54. The device, apparatus, system or method of any prior embodiments,wherein the heating zone has a surface that has an average lightabsorptance of a value given above by averaging over a wavelength range400 nm to 800 nm, 700 nm to 1,500 nm, 900 nm to 2,000 nm, or 2,000 nm to20,000 nm.

NN55. The device, apparatus, system or method of any prior embodiments,wherein the LVS ratio for sample is 5, 10, 20, 50, 70, 100, 200, 300,400, 500, 600, 700, 800, 800, 1,000, 2,000, 5,000, 10,000, 100,000, orin a range between any of the two values.

NN56. The device, apparatus, system or method of any prior embodiments,wherein the LVS ratio for sample is in a range of 5 to 10, 10 to 50, 50to 100, 100 to 500, 500 to 1,000, 1,000, to 10,000, or 10,000 to100,000,

NN57. The device, apparatus, system or method of any prior embodiments,wherein the sample has a lateral dimension of 15 mm and a thickness of30 um, hence an LVS for the sample of 500.

NN58. The device, apparatus, system or method of any prior embodiments,wherein the thickness of the relevant sample is reduced (which also canhelp sample heating speed), and the relevant sample has a thickness of0.05 um, 0.1 um, 0.2 um, 0.5 um, 1 um, 2 um, 5 um, 10 um, 20 um, 30 um,40 um, 50 um, 60 um, 70 um, 80 um, 90 um, 100 um, 200 um, 300 um, or ina range between any of the two values.

NN59. The device, apparatus, system or method of any prior embodiments,wherein the relevant sample has a thickness in a range between 0.05 umand 0.5 um, 0.5 um and 1 um, 1 um and 5 um, 5 um and 10 um, 10 um and 30um, 30 um and 50 um, 50 um and 70 um, 70 um and 100 um, 100 um and 200um, or 200 um and 300 um.

NN60. The device, apparatus, system or method of any prior embodiments,wherein the KC ratio materials for the cooling layer is equal to orhigher than 0.1 cm²/sec, 0.2 cm²/sec, 0.3 cm²/sec, 0.4 cm²/sec, 0.5cm²/sec, 0.6 cm²/sec, 0.7 cm²/sec, 0.8 cm²/sec, 0.9 cm²/sec, 1 cm²/sec,1.1 cm²/sec, 1.2 cm²/sec, 1.3 cm²/sec, 1.4 cm²/sec, 1.5 cm²/sec, 1.6cm²/sec, 2 cm²/sec, 3 cm²/sec, or in a range between any of the twovalues.

NN61. The device, apparatus, system or method of any prior embodiments,wherein the KC ratio for the cooling layer is in a range of between 0.5cm²/sec and 0.7 cm²/sec, 0.7 cm²/sec and 0.9 cm²/sec, 0.9 cm²/sec and 1cm²/sec, 1 cm²/sec and 1.1 cm²/sec, 1.1 cm²/sec and 1.3 cm²/sec, 1.3cm²/sec and 1.6 cm²/sec.

NN62. The device, apparatus, system or method of any prior embodiments,wherein a high thermal conductivity (i.e. high-K) material is used forthe cooling layer, and the high-K material has a thermal conductivity ofequal to or larger than 50 W/(m·K), 80 W/(m·K), 100 W/(m·K), 150W/(m·K), 200 W/(m·K), 250 W/(m·K), 300 W/(m·K), 350 W/(m·K), 400W/(m·K), 450 W/(m·K), 500 W/(m·K), or in a range between any of the twovalues.

NN63. The device, apparatus, system or method of any prior embodiments,wherein the sample to non-sample thermal mass ratio (NSTM ratio) is 0.1,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 1, 1.5, 2, 3, 4, 5, 10, 20, 30, 40, 50,60, 70, 100, 200, 300, 1,000, 4,000, or in a range between any of thetwo values.

NN64. The device, apparatus, system or method of any prior embodiments,wherein the sample to non-sample thermal mass ratio (NSTM ratio) is in arange of between 0.1 to 0.2, 0.2 to 0.5, 0.5 to 0.7, 0.7 to 1, 1 to 1.5,1.5 to 5, 5 to 10, 10 to 30, 30 to 50, 50 to 100, 100 to 300, 300 to1,000, or 1,000 to 4,000.

NN65. The device, apparatus, system or method of any prior embodiments,wherein the device is configured to make the sample to non-samplethermal mass ratio high, one need to keep the area thermal mass of thenon-sample low, which in turn, needs to make the plates and theheating/cooling layer thin, and/or the volume specific heat low.

NN66. The device, apparatus, system or method of any prior embodiments,wherein the device comprises a thin material that has multi-layers ormixed materials. For examples, a carbon fiber layer(s) with plasticsheets or carbon mixed with plastics, which can have a thickness of 0.1um, 0.2 um, 0.5 um, 1 um, 2 um, 5 um, 10 um, 25 um, 50 um, or in a rangebetween any of the two values.

NN67. The device, apparatus, system or method of any prior embodiments,wherein the relevant volume of the sample is 0.001 ul, 0.005 ul, 0.01ul, 0.02 ul, 0.05 ul, 0.1 ul, 0.2 ul, 0.5 ul, 1 ul, 2 ul, 5 ul, 10 ul,20 ul, 30 uL, 50 ul, 100 ul, 200 ul, 500 ul, 1 ml, 2 ml, 5 ml, or in arange between any of the two values.

NN68. The device, apparatus, system or method of any prior embodiments,wherein the relevant sample volume is in a range of 0.001 uL to 0.1 uL,0.1 um to 2 uL, 2 uL to 10 uL, 10 uL to 30 uL, 30 uL to 100 uL, 100 uLto 200 uL, or 200 uL to 1 mL.

NN69. The device, apparatus, system or method of any prior embodiments,wherein the relevant sample volume is in a range of 0.001 uL to 0.1 uL,0.1 um to 1 uL, 0.1 uL to 5 uL, or 0.1 uL to 10 uL.

NN70. The device, apparatus, system or method of any prior embodiments,wherein the ratio of the relevant sample to entire sample volume (REratio) is 0.01%, 0.05%, 0.1%, 0.5%, 0.1%, 0.5%, 1%, 5%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 100%, or in a range between any of the twovalues.

NN71. The device, apparatus, system or method of any prior embodiments,wherein the RE ratio is in a range of between 0.01% and 0.1%, 0.1% and1%, 1% and 10%, 10% and 30%, 30% and 60%, 60% and 90%, or 90% and 100%.

NN72. The device, apparatus, system or method of any prior embodiments,wherein the area of the heating zone is only a fraction of the samplelateral area, and the fraction (i.e. the ratio of the heating zone tothe sample lateral area) is 0.01%, 0.05%, 0.1%, 0.5%, 0.1%, 0.5%, 1%,5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or in a rangebetween any of the two values.

NN73. The device, apparatus, system or method of any prior embodiments,wherein the ratio of the heating zone area to the sample lateral area isin a range of between 0.01% and 0.1%, 0.1% and 1%, 1% and 10%, 10% and30%, 30% and 60%, 60% and 90%, or 90% and 99%.

NN74. The device, apparatus, system or method of any prior embodiments,wherein the scaled thermal conduction ratio (STM ratio) is 2 or larger,5 or larger, 10 or larger, 20 or larger, 30 or larger, 40 or larger, 50or larger, 100 or larger, 1,000 or larger, 10,000 or larger, 10,000 orlarger, or in a range between any of the two values.

NN75.1 The device, apparatus, system or method of any prior embodiments,wherein the scaled thermal conduction ratio (STM ratio) is in a range ofbetween 10 to 20, 30 to 50, 50 to 70, 70 to 100, 100 to 1,000, 1,000 to10,000, or 10,000 to 1,000,000.

NN75.2 The device, apparatus, system or method of any prior embodiments,wherein the scaled thermal conduction ratio (STM ratio) is in a range ofbetween 10 to 20, 30 to 50, 50 to 70, 70 to 100, 100 to 1,000, 1,000 to10,000, or 10,000 to 1,000,000; and the cooling zone (layer) has thermalconductivity times its thickness of 6×10⁻⁵ W/K, 9×10⁻⁵ W/K, 1.2×10⁻⁴W/K, 1.5×10⁻⁴ W/K, 1.8×10⁻⁴ W/K, 2.1×10⁻⁴ W/K, 2.7×10⁻⁴ W/K, 3×10⁻⁴ W/K,1.5×10⁻⁴ W/K, or in a range between any of the two values.

NN75.3 The device, apparatus, system or method of any prior embodiments,wherein the scaled thermal conduction ratio (STM ratio) is in a range ofbetween 20 to 80.

NN76. The device, apparatus, system or method of any prior embodiments,wherein the lateral to vertical size (LVS) ratio for relevant sample is5, 10, 20, 50, 70, 100, 200, 300, 400, 500, 600, 700, 800, 800, 1,000,2000, 5000, 10,000, 100,000, or in a range between any of the twovalues.

NN77. The device, apparatus, system or method of any prior embodiments,wherein the LVS ratio for relevant sample is in a range of 5 to 10, 10to 50, 50 to 100, 100 to 500, 500 to 1,000, 1,000, to 10,000, or 10,000to 100,000.

NN78. The device, apparatus, system or method of any prior embodiments,wherein the thickness of the relevant sample is reduced (which also canhelp sample heating speed), and the relevant sample has a thickness of0.05 um, 0.1 um, 0.2 um, 0.5 um, 1 um, 2 um, 5 um, 10 um, 20 um, 30 um,40 um, 50 um, 60 um, 70 um, 80 um, 90 um, 100 um, 200 um, 300 um, or ina range between any of the two values.

NN78. The device, apparatus, system or method of any prior embodiments,wherein the relevant sample has a thickness in a range between 0.05 umand 0.5 um, 0.5 um and 1 um, 1 um and 5 um, 5 um and 10 um, 10 um and 30um, 30 um and 50 um, 50 um and 70 um, 70 um and 100 um, 100 um and 200um, or 200 um and 300 um.

OO1. A device, comprising:

a first plate comprising a polymer material and having a thickness lessthan or equal to 100 μm;

a second plate comprising a polymer material and having a thickness lessthan or equal to 100 μm; and

a heating/cooling layer disposed on either the first plate or the secondplate, the heating/cooling layer having a thermal conductivity between6×10⁻⁵ W/K multiplied by the thickness of the heating/cooling layer and1.5×10⁻⁴ W/K multiplied by the thickness of the heating/cooling layer,

wherein the first plate and the second plate face each other in aparallel arrangement, and are separated from each other by a distance,and wherein the first plate and the second plate are configured toreceive a fluid sample sandwiched between the first plate and the secondplate.

OO2. A device, comprising:

a first plate;

a second plate having a thickness less than or equal to 100 μm, whereinthe second plate is separated from the first plate in a parallelarrangement by a distance less than or equal to the thickness of thesecond plate; and

a heating/cooling layer disposed on either the first plate or the secondplate,

wherein the heating/cooling layer is configured to receiveelectromagnetic radiation such that at least a portion of a liquidsample sandwiched between the first plate and the second plate is heatedat a rate of at least 30° C./sec.

OO3. A device, comprising:

a first plate;

a second plate having a thickness less than or equal to 100 μm, whereinthe second plate is separated from the first plate in a parallelarrangement by a distance less than or equal to the thickness of thesecond plate; and

a heating/cooling layer disposed on either the first plate or the secondplate,

wherein at least a portion of a liquid sample sandwiched between thefirst plate and the second plate is cooled at a rate of at least 30°C./sec when the heating/cooling layer is not receiving electromagneticradiation generated by an optical source.

OO4. A device, comprising:

a first plate;

a second plate having a thickness less than or equal to 100 μm, whereinan inner surface of the second plate is separated from an inner surfaceof the first plate in a parallel arrangement by a distance less than orequal to the thickness of the second plate;

a heating/cooling layer disposed on the inner surface or on an outersurface of the second plate; and

a layer of reagents dried on the inner surface of the first plate.

OO5. The device of any of OO1-OO4 embodiments, further comprising alight absorbing layer disposed on the heating/cooling layer, wherein thelight absorbing layer has an average light absorptance of at least 30%.

OO6. The device of OO5, wherein the light absorbing layer comprisesblack paint.

OO7. The device of any of OO1-OO6 embodiments, wherein the first plateis movable relative to the second plate.

OO8. The device of any of OO1-OO7 embodiments, wherein a thickness ofthe heating/cooling layer is less than or equal to 3 μm.

OO9. The device of any of OO1-OO8 embodiments, wherein at least one ofthe first plate and the second plate has an area across its majorsurface of about 400 mm².

OO10. The device of any of OO1-OO9 embodiments, further comprising aplurality of spherical spacers disposed between the first plate and thesecond plate.

OO11. The device of any of OO1-OO9 embodiments, further comprising aplurality of spacers having a height of about 10 um, wherein theplurality of spacers are disposed between the first plate and the secondplate.

OO12. The device of any of OO1-OO11 embodiments, wherein the distancebetween the first plate and the second plate is less than or equal to100 μm.

OO13. The device of any of OO1-OO12 embodiments, further comprising ahinge configured to connect the first plate with the second plate, andcoupled to an edge of the first plate or the second plate.

OO14. The device of any of OO1-OO13 embodiments, wherein the at least aportion of the liquid sample comprises a volume of the sample along apath of the electromagnetic radiation.

OO15. The device of any of OO1-OO14 embodiments, wherein the at least aportion of the liquid sample comprises a volume of the sample that isadjacent to the heating/cooling layer.

OO16. The device of OO4, wherein the layer of dried reagents comprisesreagents used for nucleic acid amplification.

PP1. A system, comprising:

a device, comprising:

a first plate comprising a polymer material and having a thickness lessthan or equal to 100 μm,

a second plate comprising a polymer material and having a thickness lessthan or equal to 100 μm, wherein the second plate is separated from thefirst plate in a parallel arrangement by a distance less than or equalto the thickness of the second plate,

a heating/cooling layer disposed on either the first plate or the secondplate, the heating/cooling layer having a thickness and a thermalconductivity between 6×10⁻⁵ W/K multiplied by the thickness of theheating/cooling layer and 1.5×10⁻⁴ W/K multiplied by the thickness ofthe heating/cooling layer, and

a support frame configured to support at least one of the first plateand the second plate;

a housing having a first opening configured to receive the device and atleast one other opening;

an optical source configured to direct electromagnetic radiation towardsthe heating/cooling layer,

wherein the heating/cooling layer is configured to absorb at least aportion of the electromagnetic radiation such that at least a portion ofa liquid sample sandwiched between the first plate and the second plateis heated at a rate of at least 30° C./sec, and

wherein at least the portion of the liquid sample sandwiched between thefirst plate and the second plate is cooled at a rate of at least 30°C./sec when the heating/cooling layer is not receiving theelectromagnetic radiation generated by the optical source, and whereinthe system consumes less than 500 mW of power.

PP2. A system, comprising:

a device, comprising:

a first plate,

a second plate having a thickness less than or equal to 100 μm, whereinthe second plate is separated from the first plate in a parallelarrangement by a distance less than or equal to the thickness of thesecond plate,

a heating/cooling layer disposed on either the first plate or the secondplate, and

a support frame configured to support at least one of the first plateand the second plate; and

an optical source configured to direct electromagnetic radiation towardsthe heating/cooling layer,

wherein at least a portion of a liquid sample sandwiched between thefirst plate and the second plate is cooled at a rate of at least 30°C./sec when the heating/cooling layer is not receiving theelectromagnetic radiation generated by the optical source.

PP3. A system, comprising:

a device, comprising:

a first plate,

a second plate having a thickness less than or equal to 100 μm, whereinthe second plate is separated from the first plate in a parallelarrangement by a distance less than or equal to the thickness of thesecond plate,

a heating/cooling layer disposed on either the first plate or the secondplate; and

an optical source configured to direct electromagnetic radiation towardsthe heating/cooling layer, wherein the system consumes less than 500 mWof power.

PP4. A system, comprising:

a device, comprising:

a first plate,

a second plate having a thickness less than or equal to 100 μm, whereinthe second plate is separated from the first plate in a parallelarrangement by a distance less than or equal to the thickness of thesecond plate,

a heating/cooling layer disposed on either the first plate or the secondplate, and

a support frame configured to support at least one of the first plateand the second plate;

a housing having a first opening configured to receive the device and atleast one other opening; and

an optical source configured to direct electromagnetic radiation throughthe at least one other opening of the housing and towards theheating/cooling layer,

wherein a liquid sample sandwiched between the first plate and thesecond plate is cooled at a rate of at least 30° C./sec when theheating/cooling layer is not receiving the electromagnetic radiationgenerated by the optical source.

PP5. The system of any one of PP1-PP4 embodiments, wherein the devicefurther comprises a light absorbing layer disposed on theheating/cooling layer, wherein the light absorbing layer has an averagelight absorptance of at least 30%.

PP6. The system of PP5, wherein the light absorbing layer comprisesblack paint.

PP7. The system of any one of PP1-PP6 embodiments, wherein the firstplate is movable relative to the second plate.

PP8. The system of any one of PP1-PP7 embodiments, wherein a thicknessof the heating/cooling layer is less than or equal to 3 μm.

PP9. The system of any one of PP1-PP8 embodiments, wherein at least oneof the first plate and the second plate has an area across its majorsurface of about 400 mm².

PP10. The system of any one of PP1-PP9 embodiments, wherein the opticalsource comprises a light emitting diode (LED.)

PP11. The system of PP10, wherein the LED comprises a blue LED.

PP12. The system of any one of PP1-PP11 embodiments, further comprisingan optical pipe configured to guide the electromagnetic radiation fromthe optical source to the heating/cooling layer.

PP13. The system of PP1 or PP4, wherein the at least one other openingof the housing is configured to be aligned over at least the portion ofthe liquid sample sandwiched between the first plate and the secondplate when the device is placed within the housing via the firstopening.

PP14. The system of any one of PP1-PP13 embodiments, wherein the supportframe is configured to support at least the first plate or the secondplate along a perimeter of the first plate or second plate.

QQ1. A method of using a device, comprising:

placing a second plate over a first plate such that a fluidic sample issandwiched between the first plate and the second plate at a thicknessdetermined by one or more spacers located on at least one of the firstplate and the second plate;

activating a heat source configured to radiate electromagnetic radiationtowards a heating layer located on either the first plate or the secondplate; and

heating, using at least the heating layer, at least a portion of thefluidic sample at a rate of at least 30° C./sec.

QQ2. A method of using a device, comprising:

placing a second plate over the first plate such that a fluidic sampleis sandwiched between the first plate and the second plate at athickness determined by one or more spacers located on at least one ofthe first plate and the second plate;

activating, for a given time period, a heat source configured to radiateelectromagnetic radiation towards a heating/cooling layer located oneither the first plate or the second plate;

deactivating the heat source after the given time period, wherein atleast a portion of the fluidic sample cools at a rate of at least 30°C./sec after the deactivating.

QQ3. A method of using a device, comprising:

placing a second plate over the first plate such that a fluidic sampleis sandwiched between the first plate and the second plate at athickness determined by one or more spacers located on at least one ofthe first plate and the second plate;

activating a heat source configured to radiate electromagnetic radiationtowards a heating layer located on either the first plate or the secondplate, wherein the heat source consumes less than 500 mW of power; and

heating, using at least the heating layer, at least a portion of thefluidic sample.

QQ4. The method of any one of QQ1-QQ3 embodiments, wherein the firstplate or the second plate further comprises a light absorbing layerdisposed on the heating layer, wherein the light absorbing layer has anaverage light absorptance of at least 30%.

QQ5. The method of QQ4, wherein the light absorbing layer comprisesblack paint.

QQ6. The method of any one of QQ1-QQ5 embodiments, further comprisingclosing the second plate over the first plate using a hinge connectedbetween the first plate and the second plate.

QQ7. The method of any one of QQ1-QQ6 embodiments, wherein a thicknessof the heating layer is less than or equal to 3 μm.

QQ8. The method of any one of QQ1-QQ7 embodiments, wherein at least oneof the first plate and the second plate has an area across its majorsurface of about 400 mm².

QQ9. The method of any one of QQ1-QQ8 embodiments, wherein activating aheat source comprises activating an LED to radiate light towards theheating layer.

QQ10. The method of QQ9, further comprising controlling an output of theLED based on a measured or estimated temperature of the portion of thefluidic sample.

QQ11. The method of any one of QQ1-QQ10 embodiments, further comprisingexpanding the electromagnetic radiation using a beam expander before theelectromagnetic radiation reaches the heating layer.

QQ12. The method of any one of QQ1-QQ11 embodiments, further comprisingsupporting a perimeter of either the first plate or the second plate ona support frame.

RR1. A method of amplifying nucleic acids, comprising:

depositing a fluidic sample containing nucleic acids on a first plate ofa fluidic device;

placing a second plate over the first plate such that the fluidic sampleis sandwiched between the first plate and the second plate, whereinreagents for nucleic acid amplification are present on the inner surfaceof the second plate;

activating a heat source configured to radiate electromagnetic radiationtowards a heating layer located on either the first plate or the secondplate;

heating, using at least the heating layer, at least a portion of thefluidic sample at a rate of at least 30° C./sec; and

accumulating nucleic acid amplification products in at least the portionof the fluidic sample sandwiched between the first plate and the secondplate.

RR2. A method of amplifying nucleic acids, comprising:

depositing a fluidic sample containing nucleic acids on a first plate ofa fluidic device;

placing a second plate over the first plate such that the fluidic sampleis sandwiched between the first plate and the second plate, whereinreagents for nucleic acid amplification are present on the inner surfaceof the second plate;

amplifying nucleic acids in the sample by conducting one or more PCRcycles, wherein each PCR cycle comprises a denaturing step, an annealingstep, and an elongation step;

wherein one or more of the denaturing step, the annealing step, and/orthe elongation step comprises:

activating a heat source configured to radiate electromagnetic radiationtowards a heating layer located on either the first plate or the secondplate; and

heating, using at least the heating layer, at least a portion of thefluidic sample at a rate of at least 30° C./sec.

RR3. A method of amplifying nucleic acids, comprising:

depositing a fluidic sample containing nucleic acids on a first plate ofa fluidic device;

placing a second plate over the first plate such that the fluidic sampleis sandwiched between the first plate and the second plate, whereinreagents for nucleic acid amplification are present on the inner surfaceof the second plate;

activating, for a given time period, a heat source configured to radiateelectromagnetic radiation towards a heating/cooling layer located oneither the first plate or the second plate;

deactivating the heat source after the given time period, wherein atleast a portion of the fluidic sample adjacent to the heating/coolinglayer cools at a rate of at least 30° C./sec after the deactivating; and

accumulating nucleic acid amplification products in at least the portionof the fluidic sample sandwiched between the first plate and the secondplate.

RR4. A method of amplifying nucleic acids, comprising:

depositing a fluidic sample containing nucleic acids on a first plate ofa fluidic device;

placing a second plate over the first plate such that the fluidic sampleis sandwiched between the first plate and the second plate, whereinreagents for nucleic acid amplification are present on the inner surfaceof the second plate;

amplifying nucleic acids in the sample by conducting one or more PCRcycles, wherein each PCR cycle comprises a denaturing step, an annealingstep, and an elongation step;

wherein one or more of the denaturing steps, the annealing step, and/orthe elongation step comprises:

activating a heat source configured to radiate electromagnetic radiationtowards a heating layer located on either the first plate or the secondplate; and

deactivating the heat source after the given time period, wherein atleast a portion of the fluidic sample adjacent to the heating/coolinglayer cools at a rate of at least 30° C./sec after the deactivating; and

accumulating nucleic acid amplification products in at least the portionof the fluidic sample sandwiched between the first plate and the secondplate.

RR5. A method of amplifying nucleic acids, comprising:

depositing a fluidic sample containing nucleic acids on a first plate ofa fluidic device;

placing a second plate over the first plate such that the fluidic sampleis sandwiched between the first plate and the second plate at athickness determined by one or more spacers located on at least one ofthe first plate and the second plate, wherein reagents for nucleic acidamplification are present on the inner surface of the second plate;

activating a heat source configured to radiate electromagnetic radiationtowards a heating layer located on either the first plate or the secondplate, wherein the heat source consumes less than 500 mW of power;

heating, using at least the heating layer, at least a portion of thefluidic sample; and

accumulating nucleic acid amplification products in at least the portionof the fluidic sample sandwiched between the first plate and the secondplate.

RR6. A method of amplifying nucleic acids, comprising:

depositing a fluidic sample containing nucleic acids on a first plate ofa fluidic device;

placing a second plate over the first plate such that the fluidic sampleis sandwiched between the first plate and the second plate at athickness determined by one or more spacers located on at least one ofthe first plate and the second plate, wherein reagents for nucleic acidamplification are present on the inner surface of the second plate;

amplifying nucleic acids in the sample by conducting one or more PCRcycles, wherein each PCR cycle comprises a denaturing step, an annealingstep, and an elongation step;

wherein one or more of the denaturing step, the annealing step, and/orthe elongation step comprises:

activating a heat source configured to radiate electromagnetic radiationtowards a heating layer located on either the first plate or the secondplate, wherein the heat source consumes less than 500 mW of power;

heating, using at least the heating layer, at least a portion of thefluidic sample; and

accumulating nucleic acid amplification products in at least the portionof the fluidic sample sandwiched between the first plate and the secondplate.

RR7. The method of any one of RR1-RR6 embodiments, wherein the firstplate or the second plate further comprises a light absorbing layerdisposed on the heating/cooling layer, wherein the light absorbing layerhas an average light absorptance of at least 30%.

RR8. The method of RR7, wherein the light absorbing layer comprisesblack paint.

RR9. The method of any one of RR1-RR8 embodiments, further comprisingclosing the second plate over the first plate using a hinge connectedbetween the first plate and the second plate.

RR10. The method of any one of RR1-RR9 embodiments, wherein a thicknessof the heating/cooling layer is less than or equal to 3 μm.

RR11. The method of any one of RR1-RR10 embodiments, wherein at leastone of the first plate and the second plate has an area across its majorsurface of about 400 mm².

RR12. The method of any one of RR1-RR11 embodiments, wherein activatinga heat source comprises activating an LED to radiate light towards theheating/cooling layer.

RR13. The method of RR12, further comprising controlling an output ofthe LED based on a measured or estimated temperature of the portion ofthe fluidic sample.

RR14. The method of any one of RR1-RR13 embodiments, further comprisingexpanding the electromagnetic radiation using a beam expander before theelectromagnetic radiation reaches the heating layer.

RR15. The method of any one of RR1-RR14 embodiments, further comprisingsupporting a perimeter of either the first plate or the second plate ona support frame.

SS1. A method for detecting whether a target nucleic acid sequence ispresent or absent in a sample, comprising:

depositing a fluidic sample containing nucleic acids on a first plate ofa fluidic device;

placing a second plate over the first plate such that the fluidic sampleis sandwiched between the first plate and the second plate, whereinreagents for nucleic acid amplification are present on the inner surfaceof the second plate, and wherein the reagents comprise primers that canhybridize with the target nucleic acid;

activating a heat source configured to radiate electromagnetic radiationtowards a heating layer located on either the first plate or the secondplate;

heating, using at least the heating layer, at least a portion of thefluidic sample at a rate of at least 30° C./sec; and

detecting whether the fluidic sample contains amplified product of thetarget nucleic acid sequence.

SS2. A method for detecting whether a target nucleic acid sequence ispresent or absent in a sample, comprising:

depositing a fluidic sample containing nucleic acids on a first plate ofa fluidic device;

placing a second plate over the first plate such that the fluidic sampleis sandwiched between the first plate and the second plate, whereinreagents for nucleic acid amplification are present on the inner surfaceof the second plate, and wherein the reagents comprise primers that canhybridize with the target nucleic acid;

activating, for a given time period, a heat source configured to radiateelectromagnetic radiation towards a heating/cooling layer located oneither the first plate or the second plate;

deactivating the heat source after the given time period, wherein atleast a portion of the fluidic sample adjacent to the heating/coolinglayer cools at a rate of at least 30° C./sec after the deactivating; and

detecting whether the fluidic sample contains amplified product of thetarget nucleic acid sequence.

SS3. A method for detecting whether a target nucleic acid sequence ispresent or absent in a sample, comprising:

depositing a fluidic sample containing nucleic acids on a first plate ofa fluidic device;

placing a second plate over the first plate such that the fluidic sampleis sandwiched between the first plate and the second plate, whereinreagents for nucleic acid amplification are present on the inner surfaceof the second plate, and wherein the reagents comprise primers that canhybridize with the target nucleic acid;

activating a heat source configured to radiate electromagnetic radiationtowards a heating layer located on either the first plate or the secondplate, wherein the heat source consumes less than 500 mW of power;

heating, using at least the heating layer, at least a portion of thefluidic sample; and

detecting whether the fluidic sample contains amplified product of thetarget nucleic acid sequence.

SS4. The method of any one of SS1-SS3 embodiments, wherein the firstplate or the second plate further comprises a light absorbing layerdisposed on the heating/cooling layer, wherein the light absorbing layerhas an average light absorptance of at least 30%.

SS5. The method of SS4, wherein the light absorbing layer comprisesblack paint.

SS6. The method of any one of SS1-SS5 embodiments, further comprisingclosing the second plate over the first plate using a hinge connectedbetween the first plate and the second plate.

SS7. The method of any one of SS1-SS6 embodiments, wherein a thicknessof the heating/cooling layer is less than or equal to 3 μm.

SS8. The method of any one of SS1-SS7 embodiments, wherein at least oneof the first plate and the second plate has an area across its majorsurface of about 400 mm².

SS9. The method of any one of SS1-SS8 embodiments, wherein activating aheat source comprises activating an LED to radiate light towards theheating/cooling layer.

SS10. The method of SS9, further comprising controlling an output of theLED based on a measured or estimated temperature of the portion of thefluidic sample.

SS11. The method of any one of SS1-SS10 embodiments, further comprisingexpanding the electromagnetic radiation using a beam expander before theelectromagnetic radiation reaches the heating layer.

SS12. The method of any one of SS1-SS11 embodiments, further comprisingsupporting a perimeter of either the first plate or the second plate ona support frame.

TT1. A method for detecting the presence or absence of an analyte in asample, comprising:

depositing a fluidic sample on a first plate of a fluidic device;

placing a second plate over the first plate such that the fluidic sampleis sandwiched between the first plate and the second plate, whereinreagents for detection of the analyte are present on the inner surfaceof the second plate;

activating a heat source configured to radiate electromagnetic radiationtowards a heating layer located on either the first plate or the secondplate;

heating, using at least the heating layer, at least a portion of thefluidic sample at a rate of at least 30° C./sec; and

detecting whether the fluidic sample contains the analyte.

TT2. A method for detecting the presence or absence of an analyte in asample, comprising:

depositing a fluidic sample containing on a first plate of a fluidicdevice;

placing a second plate over the first plate such that the fluidic sampleis sandwiched between the first plate and the second plate, whereinreagents for detection of the analyte are present on the inner surfaceof the second plate;

activating, for a given time period, a heat source configured to radiateelectromagnetic radiation towards a heating/cooling layer located oneither the first plate or the second plate;

deactivating the heat source after the given time period, wherein atleast a portion of the fluidic sample adjacent to the heating/coolinglayer cools at a rate of at least 30° C./sec after the deactivating; and

detecting whether the fluidic sample contains the analyte.

TT3. A method for detecting the presence or absence of an analyte in asample, comprising:

depositing a fluidic sample on a first plate of a fluidic device;

placing a second plate over the first plate such that the fluidic sampleis sandwiched between the first plate and the second plate, whereinreagents for detection of the analyte are present on the inner surfaceof the second plate;

activating a heat source configured to radiate electromagnetic radiationtowards a heating layer located on either the first plate or the secondplate, wherein the heat source consumes less than 500 mW of power;

heating, using at least the heating layer, at least a portion of thefluidic sample; and

detecting whether the fluidic sample contains the analyte.

UU1. A method for diagnosing a condition in a subject, comprising:

depositing a fluidic sample from the subject on a first plate of afluidic device;

placing a second plate over the first plate such that the fluidic sampleis sandwiched between the first plate and the second plate, whereinreagents for detection of an analyte are present on the inner surface ofthe second plate;

activating a heat source configured to radiate electromagnetic radiationtowards a heating layer located on either the first plate or the secondplate;

heating, using at least the heating layer, at least a portion of thefluidic sample at a rate of at least 30° C./sec; and

detecting whether the fluidic sample contains the analyte;

wherein presence or absence of the analyte indicates that the subjecthas the condition.

UU2. A method for diagnosing a condition in a subject, comprising:

depositing a fluidic sample from the subject on a first plate of afluidic device;

placing a second plate over the first plate such that the fluidic sampleis sandwiched between the first plate and the second plate, whereinreagents for detection of the analyte are present on the inner surfaceof the second plate;

activating, for a given time period, a heat source configured to radiateelectromagnetic radiation towards a heating/cooling layer located oneither the first plate or the second plate;

deactivating the heat source after the given time period, wherein atleast a portion of the fluidic sample adjacent to the heating/coolinglayer cools at a rate of at least 30° C./sec after the deactivating; and

detecting whether the fluidic sample contains the analyte;

wherein presence or absence of the analyte indicates that the subjecthas the condition.

UU3. A method for diagnosing a condition in a subject, comprising:

depositing a fluidic sample from the subject on a first plate of afluidic device;

placing a second plate over the first plate such that the fluidic sampleis sandwiched between the first plate and the second plate, whereinreagents for detection of an analyte are present on the inner surface ofthe second plate;

activating a heat source configured to radiate electromagnetic radiationtowards a heating layer located on either the first plate or the secondplate, wherein the heat source consumes less than 500 mW of power;

heating, using at least the heating layer, at least a portion of thefluidic sample; and

detecting whether the fluidic sample contains the analyte;

wherein presence or absence of the analyte indicates that the subjecthas the condition.

UU4. A method for diagnosing a condition in a subject, comprising:

depositing a fluidic sample from the subject on a first plate of afluidic device;

placing a second plate over the first plate such that the fluidic sampleis sandwiched between the first plate and the second plate, whereinreagents for detection of an analyte are present on the inner surface ofthe second plate;

activating a heat source configured to radiate electromagnetic radiationtowards a heating layer located on either the first plate or the secondplate;

heating, using at least the heating layer, at least a portion of thefluidic sample at a rate of at least 30° C./sec;

quantifying the amount of the analyte in the fluidic sample; and

comparing the amount to a control or reference amount of the analyte;

wherein a greater or reduced amount of the analyte in the samplecompared to the control or reference amount indicates that the subjecthas the condition.

UU5. A method for diagnosing a condition in a subject, comprising:

depositing a fluidic sample from the subject on a first plate of afluidic device;

placing a second plate over the first plate such that the fluidic sampleis sandwiched between the first plate and the second plate, whereinreagents for detection of an analyte are present on the inner surface ofthe second plate;

activating, for a given time period, a heat source configured to radiateelectromagnetic radiation towards a heating/cooling layer located oneither the first plate or the second plate;

deactivating the heat source after the given time period, wherein atleast a portion of the fluidic sample adjacent to the heating/coolinglayer cools at a rate of at least 30° C./sec after the deactivating;

quantifying the amount of the analyte in the fluidic sample; and

comparing the amount to a control or reference amount of the analyte;

wherein a greater or reduced amount of the analyte in the samplecompared to the control or reference amount indicates that the subjecthas the condition.

UU6. A method for diagnosing a condition in a subject, comprising:

depositing a fluidic sample from the subject on a first plate of afluidic device;

placing a second plate over the first plate such that the fluidic sampleis sandwiched between the first plate and the second plate, whereinreagents for detection of an analyte are present on the inner surface ofthe second plate;

activating a heat source configured to radiate electromagnetic radiationtowards a heating layer located on either the first plate or the secondplate, wherein the heat source consumes less than 500 mW of power;

heating, using at least the heating layer, at least a portion of thefluidic sample; and

quantifying the amount of the analyte in the fluidic sample; and

comparing the amount to a control or reference amount of the analyte;

wherein a greater or reduced amount of the analyte in the samplecompared to the control or reference amount indicates that the subjecthas the condition.

VV1. A kit, comprising:

a device of any one of OO embodiments; and

a pre-mixed polymerase chain reaction medium.

VV2. The kit of VV1, wherein the pre-mixed polymerase chain reactionmedium comprises: a DNA template, two primers, a DNA polymerase,deoxynucleoside triphosphates (dNTPs), a bivalent cation, a monovalentcation, and a buffer solution.

PCR and Molecule Amplification

In some embodiments, the device, apparatus, system, and/or method hereindescribed can be used for rapid molecule (e.g. nucleic acid)amplification. In certain embodiments, the device, apparatus, system,and method can be used for isothermal nucleic acid amplification. Incertain embodiments, the device, apparatus, and system can be used fornon-isothermal nucleic acid amplification.

Non-isothermal nucleic acid amplification generally requires the cycledaddition and removal of thermal energy. Many non-isothermal strategiesthat can be used for nucleic acid amplification involve the heating andcooling, to precise temperatures at precise times, of a reaction mixturethat includes one or several nucleic acids of interest (that can orcannot be chemically modified with additional agents) and reagentsnecessary to complete an amplification reaction. Non-limiting examplesof such nucleic acid amplification reactions include PCR; variants ofPCR (e.g., reverse transcriptase PCR (RT-PCR), quantitative PCR (Q-PCR),or realtime quantitative PCR (RTQ-PCR)); ligase-chain reaction (LCR);variants of LCR (e.g., reverse transcriptase LCR (RTLCR), quantitativeLCR (Q-LCR), real-time quantitative LCR (RTQ-LCR)); and digital nucleicamplification reactions (e.g., digital PCR (dPCR), digital RT-PCR(dRT-PCR), digital Q-PCR (dQ-PCR), digital RTQ-PCR (dRTQ-PCR), digitalLCR (dLCR), digital RT-LCR (dRT-LCR), digital Q-LCR (dQ-LCR), digitalRTQ-LCR (dRTQ-LCR). These nucleic acid amplification reactions, andothers, are described in more detail below.

PCR Reactions

The device, apparatus, system, and method of the current disclosure caninclude the completion of a PCR amplification reaction, or any stepcomprising a PCR amplification (e.g., denaturation, annealing,elongation, etc). In some embodiments, a sample can comprise reagentsnecessary to complete a PCR reaction. Nonlimiting examples of reagentsfor a PCR reaction include a template nucleic acid (e.g., DNA) moleculeto be amplified, a set of two primers that can hybridize with a targetsequence on the template nucleic acid, a polymerase (e.g., DNApolymerase), deoxynucleotide triphosphates (dNTPs), a buffer at a pH andconcentration suitable for a desired PCR reaction, a monovalent cation,and a divalent cation. Generally, the ratio of each reagent in thesample can vary and depend upon, for example, the amount of nucleic acidto be amplified and/or the desired amount of amplification products.Methods to determine the ratio of each reagent necessary for a PCRamplification reaction are found in, for example, U.S. Pat. Nos.4,683,202 and 4,683,195, which are entirely incorporated herein byreference for all purposes.

PCR generally involves the heating and cooling of a reaction mixturethat includes several key reagents and a nucleic acid (e.g., DNA)template. Non-limiting examples of reagents that, in addition to anucleic acid template, can be used for PCR include primers, apolymerase, deoxynucleoside triphosphates (dNTPs), buffer solution,divalent cations, and monovalent cations. In general, at least twodifferent primers per nucleic acid template can be included in thereaction mixture, wherein each primer is complementary to a portion of(e.g., the 3′ ends of) the nucleic acid template. The nucleic acidtemplate is replicated by a polymerase.

Non-limiting examples of DNA polymerases that can be useful in PCRinclude Taq polymerase, Pfu polymerase, Pwo polymerase, Tfl polymerase,rTth polymerase, Tli polymerase, Tma polymerase, and VentR polymerase,Kapa2g polymerase, KOD polymerase, HaqZ05 polymerase, Haqz05 polymerase,or combinations thereof.

dNTPs are nucleotides that include triphosphate groups and are generallythe building-blocks from which amplified DNA is synthesized.Non-limiting examples of dNTPs useful in PCR include deoxyadenosinetriphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidinetriphosphate (dCTP), and deoxythymidine triphosphate (dTTP).

A buffer solution can be generally used to provide a suitable chemicalenvironment (e.g., pH, ionic strength, etc.) for optimum activity andstability of the DNA polymerase and/or other dependent components in thereaction mixture. For example, buffers of Tris-hydrochloride can beuseful in PCR methods.

Divalent cations can also be required for DNA polymerase functionality,with non-limiting examples including magnesium ions (Mg²⁺) and manganese(Mn²⁺) ions. Monovalent cations, such as, for example, potassium ions(K⁺) can be included and can be useful in minimizing the production ofunwanted, non-specific amplification products.

In some embodiments, the reagents for a PCR reaction can be a componentof an assay designed to test a blood or other liquid sample for thepresence of an analyte. For example, chloride ions can be measured byany of the following protocols, and components of these assays can bepresent in a storage site: Colorimetric methods: chloride ions displacethiocyanate from mercuric thiocyanate. Free thiocyanate reacts withferric ions to form a colored complex—ferric thiocyanate, which ismeasured photometrically. Coulometric methods: passage of a constantdirect current between silver electrodes produces silver ions, whichreact with chloride, forming silver chloride. After all the chloridecombines with silver ions, free silver ions accumulate, causing anincrease in current across the electrodes and indicating the end pointto the reaction. Mercurimetric methods: chloride is titrated with astandard solution of mercuric ions and forms HgCl2 soluble complex. Theend point for the reaction is detected colorimetrically when excessmercury ions combine with an indicator dye, diphenylcarbazon, to form ablue color. Likewise, magnesium can be measured colorimetrically usingcalmagite, which turns a red-violet color upon reaction with magnesium;by a formazan dye test; emits at 600 nm upon reaction with magnesium orusing methylthymol blue, which binds with magnesium to form a bluecolored complex. Likewise, calcium can be detected by a colorimetrictechnique using O-Cresolphtalein, which turns a violet color uponreaction of O-Cresolphtalein complexone with calcium. Likewise,Bicarbonate can be tested bichromatically because bicarbonate (HCO3⁻)and phosphoenolpyruvate (PEP) are converted to oxaloacetate andphosphate in the reaction catalyzed by phosphoenolpyruvate carboxylase(PEPC). Malate dehydrogenase (MD) catalyzes the reduction ofoxaloacetate to malate with the concomitant oxidation of reducednicotinamide adenine dinucleotide (NADH). This oxidation of NADH resultsin a decrease in absorbance of the reaction mixture measuredbichromatically at 380/410 nm proportional to the Bicarbonate content ofthe sample. Blood urea nitrogen can be detected in a colorimetric testin which diacetyl, or fearon develops a yellow chromogen with urea andcan be quantified by photometry, or multiusing the enzyme urease, whichconverts urea to ammonia and carbonic acid, which can be assayed by,e.g., i) decrease in absorbance at 340 nm when the ammonia reacts withalpha-ketoglutaric acid, ii) measuring the rate of increase inconductivity of the solution in which urea is hydrolyzed. Likewise,creatinine can be measured colorimetrically, by treated the sample withalkaline picrate solution to yield a red complex. In addition, creatinecan be measured using a non-Jaffe reaction that measures ammoniagenerated when creatinine is hydrolyzed by creatinine iminohydrolase.Glucose can be measured in an assay in which blood is exposed to a fixedquantity of glucose oxidase for a finite period of time to estimateconcentration. After the specified time, excess blood is removed and thecolor is allowed to develop, which is used to estimate glucoseconcentration. For example, glucose oxidase reaction with glucose formsnascent oxygen, which converts potassium iodide (in the filter paper) toiodine, forming a brown color. The concentration of glycosylatedhemoglobin as an indirect read of the level of glucose in the blood.When hemolysates of red cells are chromatographed, three or more smallpeaks named hemoglobin A1a, A1b, and A1c are eluted before the mainhemoglobin A peak. These “fast” hemoglobins are formed by theirreversible attachment of glucose to the hemoglobin in a two-stepreaction. Hexokinase can be measured in an assay in which glucose isphosphorylated by hexokinase (HK) in the presence of adenosinetriphosphate (ATP) and magnesium ions to produce glucose-6-phosphate andadenosine diphosphate (ADP). Glucose-6-phosphate dehydrogenase (G6P-DH)specifically oxidises glucose-6-phosphate to gluconate-6-phosphate withthe concurrent reduction of NAD+ to NADH. The increase in absorbance at340 nm is proportional to the glucose concentration in the sample. HDL,LDL, triglycerides can be measured using the Abell-Kendall protocol thatinvolves color development with Liebermann-Burchard reagent (mixedreagent of acetic anhydride, glacial acetic acid, and concentratedsulfuric acid) at 620 nm after hydrolysis and extraction of cholesterol.A fluorometric analysis can be used utilized to determine triglyceridereference values. Plasma high-density lipoprotein cholesterol (HDL-C)determination is measured by the same procedures used for plasma totalcholesterol, after precipitation of apoprotein B-containing lipoproteinsin whole plasma (LDL and VLDL) by heparin-manganese chloride. Thesecompounds can also be detected colorimetrically in an assay that isbased on the enzyme driven reaction that quantifies both cholesterolesters and free cholesterol. Cholesterol esters are hydrolyzed viacholesterol esterase into cholesterol, which is then oxidized bycholesterol oxidase into the ketone cholest-4-en-3-one plus hydrogenperoxide. The hydrogen peroxide is then detected with a highly specificcolorimetric probe. Horseradish peroxidase catalyzes the reactionbetween the probe and hydrogen peroxide, which bind in a 1:1 ratio.Samples can be compared to a known concentration of cholesterolstandard.

A single cycle of PCR typically comprises a series of steps that includea denaturation step, an annealing step, and an elongation step. Duringdenaturation, a double-stranded DNA template can be melted into itsindividual strands, such that the hydrogen bonds formed between bases ineach base-pair of the double-stranded DNA are broken.

After denaturation, an annealing step is completed, wherein the reactionmixture is incubated under conditions at which the primers hybridizewith complementary sequences present on each of the original individualstrands. After annealing, the elongation step commences, wherein theprimers are extended by a DNA polymerase, using dNTPs present in thereaction mixture. At the conclusion of elongation, two newdouble-stranded DNA molecules result, each comprising one of theoriginal individual strands of the DNA template. Each step of PCR isgenerally initiated by a change in the temperature of the reactionmixture that results from the heating or cooling of the reactionmixture. At the completion of a single round of amplification, thethermal cycle can be repeated for further rounds of amplification. Thegeneration of replicate amplification products is theoreticallyexponential with each subsequent thermal cycle. For example, for asingle DNA template, each step n, can result in a total of r replicates.

Successful PCR amplification requires high yield, high selectivity, anda controlled reaction rate at each step. Yield, selectivity, andreaction rate also generally depend on temperature, and optimaltemperatures depend on the composition and length of the polynucleotide,enzymes, and other components in the reaction mixture. In addition,different temperatures can be optimal for different steps or differentnucleic acids to be amplified. Moreover, optimal reaction conditions canvary, depending on the sequence of the template DNA, sequence of adesigned primer, and composition of the reaction mixture. Thermal cydersthat can be used to perform a PCR reaction can be programmed byselecting temperatures to be maintained, time durations for each portionof a cycle, number of cycles, rate of temperature change, and the like.

Primers for PCR can be designed according to known algorithms. Forexample, algorithms implemented in commercially available or customsoftware can be used to design primers. In some examples, primers canconsist of at least about 12 bases. In other examples, a primer canconsist of at least about 15, 18, or 20 bases in length. In still otherexamples, a primer can be up to 50+ bases in length. Primers can bedesigned such that all of the primers participating in a particularreaction have melting temperatures that are within at least about 5° C.,and more typically within about 2° C. of each other. Primers can befurther designed to avoid selfhybridization or hybridization with otherdesired primers. Those of skill in the art will recognize that theamount or concentration of primer in a reaction mixture will vary, forexample, according to the binding affinity of the primers for a giventemplate DNA and/or the quantity of available template DNA. Typicalprimer concentrations, for example, can range from 0.01 μM to 0.5 μM.

In an example PCR reaction, a reaction mixture, including adouble-stranded DNA template and additional reagents necessary for PCR,is heated to about 80-98° C. and held at that temperature for about10-90 seconds, in order to denature the DNA template into its individualstrands. Each individual strand, during the annealing step, is thenhybridized to its respective primer included in the reaction mixture bycooling the reaction mixture to a temperature of about 30-65° C. andholding it at that temperature for about 1-2 minutes. The elongationstep then commences, wherein elongation of the respective primershybridized to each individual strand occurs by the action of a DNApolymerase adding dNTPs to the primers. Elongation is initiated byheating the reaction mixture to a temperature of about 70-75° C. andholding at that temperature for 30 seconds to 5 minutes. The reactioncan be repeated for any desired number of cycles depending on, forexample, the initial amount of DNA template, the length of the desiredamplification product, the amount of dNTPs, the amount of primer, and/orprimer stringency.

While general PCR methods can be useful for nucleic acid amplification,other more specialized forms of PCR can be even more useful for a givenapplication. Nonlimiting examples of commonly used, more-specializedforms of PCR include reverse transcription PCR (RT-PCR) (e.g., U.S. Pat.No. 7,883,871), quantitative PCR (qPCR) (e.g., U.S. Pat. No. 6,180,349),real-time quantitative PCR (RTQ-PCR) (e.g., U.S. Pat. No. 8,058,054),allele-specific PCR (e.g., U.S. Pat. No. 5,595,890), assembly PCR (e.g.U.S. Patent Publication No. 20120178129), asymmetric PCR (e.g., EuropeanPatent Publication No. EP23 73 807), dial-out PCR (e.g., Schwartz J,NATURE METHODS, September 2012; 9(9): 913-915), helicase-dependent PCR(e.g., Vincent M, EMBO REPORTS 5, 2004, 5(8): 795-800), hot start PCR(e.g., European Patent Publication No. EP1419275), inverse PCR (e.g.,U.S. Pat. No. 6,607,899), methylation-specific PCR (e.g., EuropeanPatent Publication No. EP1690948), miniprimer PCR (U.S. PatentPublication No. 20120264132), multiplex PCR (U.S. Patent Publication No.20120264132), nested PCR (U.S. Patent Publication No. 20120264132),overlap-extension PCR (U.S. Patent Publication No. 20120264132), thermalasymmetric interlaced PCR (U.S. Patent Publication No. 20120264132), andtouchdown PCR (U.S. Patent Publication No. 20120264132). The device,apparatus, system, and/or method herein disclosed can be utilized toconduct such more-specialized forms of PCR.

RT-PCR Reactions

The device, apparatus, system, and method of the current disclosure caninclude the completion of an RT-PCR amplification reaction, and, thus, asample can comprise reagents necessary to complete a RT-PCR reaction.Non-limiting examples of such reagents include the reagents necessary tocomplete a PCR reaction, a reverse transcriptase, and a RNA templatethat can be used to synthesize a complementary DNA (cDNA) complement. Incases where reverse transcriptase must be removed prior to cDNAamplification, a sample supplied to a thermal cycler cannot containreagents necessary to complete a PCR reaction and can require a separateamplification reaction. Generally, the ratio of each reagent in thesample can vary and depend upon, for example, the amount of nucleic acidto be amplified and/or the desired amount of amplification products.Methods to determine the ratio of each reagent necessary for an RT-PCRamplification reaction are generally known by those skilled in the art.

Reverse transcription refers to a process by which ribonucleic acid(RNA) is replicated to its single-stranded complementary DNA (cDNA) by areverse transcriptase enzyme. Non-limiting examples of reversetranscriptase enzymes include Moloney murine leukemia virus (MMLV)transcriptase, avian myeloblastosis virus (AMY) transcriptase, variantsof AMV-transcriptase, or reverse transcriptases that have endo Hactivity. In reverse transcription PCR(RT-PCR), a reverse transcriptase,generally with endo H3 activity, is added to a reaction mixture thatincludes an RNA template and necessary reagents for PCR. The reversetranscriptase can complete RNA template replication to cDNA, byhybridizing dNTPs to the RNA template at proper conditions.

At the conclusion of replication, the reverse transcriptase can removethe single-stranded, cDNA replicated from the RNA template to permitadditional replication of the cDNA with PCR methods described above. ThecDNA and its amplification products that are produced from PCR can beused indirectly to garner information about the RNA, such as, forexample, the sequence of the RNA. The cDNA product that is synthesizedfrom an RNA by a reverse transcriptase can be removed from the reactionmixture to be used as a DNA template in a separate, subsequent set ofPCR reactions or amplification via PCR can occur in situ where reversetranscriptase is included in the reaction mixture with reagentsnecessary for PCR.

Q-PCR or RTQ-PCR Reactions

The device, apparatus, system, and method of the current disclosure caninclude the completion of a Q-PCR or RTQ-PCR amplification reaction,and, thus a sample can comprise reagents necessary to complete a Q-PCRor RTQ-PCR amplification reaction. Non-limiting examples of suchreagents include the reagents necessary to complete a PCR reaction and areporter used to detect amplification products. Generally, the ratio ofeach reagent in the sample can vary and depend upon, for example, theamount of nucleic acid to be amplified and/or the desired amount ofamplification products. Methods to determine the ratio of each reagentnecessary for a Q-PCR or RTQ-PCR amplification reaction are generallyknown by those skilled in the art.

Quantitative PCR (Q-PCR) is a variation of PCR in which the amount oftemplate DNA in a sample is quantified. Generally, amplificationproducts produced by PCR methods are linked to a reporter, such as, forexample, a fluorescent dye. At the end of a reaction, the reporter canbe detected and the results back-calculated (based on the associationratio of reporter to DNA and the known number of thermal cycles) todetermine the amount of original DNA template present. In some examples,the fluorescent dye can be detected in real time as amplificationprogresses. Such a variation of Q-PCR can be appropriately calledreal-time quantitative PCR (RTQ-PCR), real-time PCR, or kinetic PCR.Both Q-PCR and RTQ-PCR can be used to determine whether or not aspecific DNA template is present in a sample. In general, due to thepossible changes to reaction efficiency as the number of PCR cyclesincreases, however, RTQ-PCR methods can be generally more sensitive,more reliable, and thus, more frequently employed by those skilled inthe art as measurements are made on amplification products as they aresynthesized rather than on the aggregate of amplification productsobtained at the completion of the desired number of thermal cycles.Q-PCR and RTQ-PCR can also be combined with other PCR methods, such as,for example, RT-PCR. As an example, utility of combining Q-PCR orRTQ-PCR with other PCR methods, reporters can be included in an RT-PCRreaction mixture to detect and/or quantify low levels of messenger RNA(mRNA) via replication of its associated cDNA, which can enable thequantification of relative gene expression in a particular cell ortissue.

One or more reporters can be used to quantify DNA amplified as part ofQ-PCR and RTQ-PCR methods. Reporters can be associated with DNA both bycovalent and/or non-covalent linkages (e.g., ionic interactions, Van derWaals forces, hydrophobic interactions, hydrogen bonding, etc.). Forexample, a fluorescent dye that non-covalently intercalates withdouble-stranded DNA can be used as a reporter. In another example, a DNAoligonucleotide probe that fluoresces when hybridized with acomplementary DNA can be used as a reporter. In some examples, reporterscan bind to initial reactants and changes in reporter levels can be usedto detect amplified DNA. In other examples, reporters can only bedetectable or non-detectable as DNA amplification progresses. Detectionof reporters can be accomplished with one of many detection systems thatare suitable in the art. Optical detectors (e.g., fluorimeters,ultra-violet/visible light absorbance spectrophotometers) orspectroscopic detectors (e.g., nuclear magnetic resonance (NMR),infrared spectroscopy) can be, for example, useful modalities ofreporter detection. Gel based techniques, such as, for example, gelelectrophoresis can also be used for detection.

A reporter used in a Q-PCR or RTQ-PCR reaction can be an intercalatorthat can be detected. An intercalator generally binds to DNA bydisrupting hydrogen bonds between complementary bases, and, instead fitsitself between the disrupted bases. An intercalator can form its ownhydrogen bonds with one or more of the disrupted bases. Non-limitingexamples of intercalators include SYBR green, SYBR blue, DAPI, propidiumiodine, Hoeste, SYBR gold, ethidium bromide, acridines, proflavine,acridine orange, acriflavine, fluorcoumanin, ellipticine, daunomycin,chloroquine, distamycin D, chromomycin, homidium, mithramycin, rutheniumpolypyridyls, anthramycin, phenanthridines and acridines, ethidiumbromide, propidium iodide, hexidium iodide, dihydroethidium, ethidiumhomodimer-1 and -2, ethidium monoazide, and ACMA.

A reporter used in a Q-PCR or RTQ-PCR reaction can be a minor groovebinder that can be detected. Nonlimiting examples of minor grove bindersinclude indoles and imidazoles (e.g., Hoechst 33258, Hoechst 33342,Hoechst 34580 and DAPI).

A reporter used in a Q-PCR or RTQ-PCR reaction can be a nucleic acidstain that can be detected. Non-limiting examples of nucleic acid stainsinclude acridine orange (also capable of intercalating), 7-AAD,actinomycin D, LDS751, hydroxystilbamidine, SYTOX Blue, SYTOX Green,SYTOX Orange, POPO-1, POPO-3, YOYO-1, YOYO-3, TOTO-I, TOTO-3, JOJO-I,LOLO-1, BOBO-1, BOBO-3, PO-PRO-1, PO-PRO-3, BO-PRO-1, BO-PRO-3,TO-PRO-1, TO-PRO-3, TO-PRO-5, JO-PRO-1, LO-PRO-1, YO-PRO-1, YO-PRO-3,PicoGreen, OliGreen, RiboGreen, SYBR Gold, SYBR Green I, SYBR Green II,SYBR DX, SYTO-40, -41, -42, -43, -44, -45 (blue), SYTO-13, -16, -24,-21, -23, -12, -11, -20, -22, -15, -14, -25 (green), SYTO-81, -80, -82,-83, -84, -85 (orange), SYTO-64, -17, -59, -61, -62, -60, -63 (red).

A reporter used in a Q-PCR or RTQ-PCR reaction can be a fluorescent dyethat can be detected. Non-limiting examples of fluorescent dyes includefluorescein, fluorescein isothiocyanate (FITC), tetramethyl rhodamineisothiocyanate (TRITC), rhodamine, tetramethyl rhodamine,R-phycoerythrin, Cy-2, Cy-3, Cy-3.5, Cy-5, Cy5.5, Cy-7, Texas Red,Phar-Red, allophycocyanin (APC), Sybr Green I, Sybr Green II, Sybr Gold,CellTracker Green, 7-AAD, ethidium homodimer I, ethidium homodimer II,ethidium homodimer III, ethidium bromide, umbelliferone, eosin, greenfluorescent protein, erythrosin, coumarin, methyl coumarin, pyrene,malachite green, stilbene, lucifer yellow, cascade blue,dichlorotriazinylamine fluorescein, dansyl chloride, fluorescentlanthanide complexes such as those including europium and terbium,carboxy tetrachloro fluorescein, 5 and/or 6-carboxy fluorescein (FAM),5-(or 6-) iodoacetamidofluorescein, 5-{[2(and3)-5-(Acetylmercapto)-succinyl]amino} fluorescein (SAMSA-fluorescein),lissamine rhodamine B sulfonyl chloride, 5 and/or 6 carboxy rhodamine(ROX), 7-aminomethyl-coumarin, 7-Amino-4-methylcoumarin-3-acetic acid(AMCA), BODIPY fluorophores, 8-methoxypyrene-1,3,6-trisulfonic acidtrisodium salt, 3,6-Disulfonate-4-aminonaphthalimide, phycobiliproteins,AlexaFluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 633, 635,647, 660, 680, 700, 750, and 790 dyes, DyLight 350, 405, 488, 550, 594,633, 650, 680, 755, and 800 dyes, or other fluorophores known to thoseof skill in the art. For detailed listing of fluorophores that can beuseful in Q-PCR and RTQ-PCR methods, see also Hermanson, G. T.,

BIOCONJUGATE TECHNIQUES (Academic Press, San Diego, 1996) and Lakowicz,J. R., PRINCIPLES OF FLUORESCENCE SPECTROSCOPY, (Plenum Pub Corp, 2ndedition (July 1999)), which are incorporated herein by reference.

A reporter used in a Q-PCR or RTQ-PCR reaction can be a radioactivespecies that can be detected. Nonlimiting examples of radioactivespecies that can be useful in Q-PCR and RTQ-PCR methods include 14C 12311241 1251 131I, Tc99m, 35S, or 3H.

A reporter used in a Q-PCR or RTQ-PCR reaction can be an enzyme that canproduce a detectable signal. Such signal can be produced by action ofthe enzyme on its given substrate. Non-limiting examples of enzymes thatcan be useful in Q-PCR or RTQ-PCR methods include alkaline phosphatase,horseradish peroxidase, I2-galactosidase, alkaline phosphatase,galactosidase, acetylcholinesterase, and luciferase.

A reporter used in a Q-PCR or RTQ-PCR reaction can be an affinityligand-label that can be detected. A particular ligand can include alabel, such as for example, a fluorescent dye, and binding of thelabeled ligand to its substrate can produce a useful signal.Non-limiting examples of binding pairs that can be useful in Q-PCR orRTQ-PCR methods include streptavidin/biotin, avidin/biotin or anantigen/antibody complex, such as, for example, rabbit IgG andanti-rabbit IgG;

A reporter used in a Q-PCR or RTQ-PCR reaction can be a nanoparticlethat can be detected via light scattering or surface plasmon resonance(SPR). Non-limiting examples of materials useful for SPR-based detectioninclude gold and silver materials. Other nanoparticles that can beuseful in Q-PCR or RTQ-PCR reactions can be quantum dots (Qdots). Qdotsare generally constructed of semiconductor nanocrystals, described, forexample in U.S. Pat. No. 6,207,392. Nonlimiting examples ofsemiconductor materials that can be used to produce a Qdot include MgS,MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, ZnS,ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe, HgTe, GaAs, InGaAs, InP, InAs,or mixed compositions thereof.

A reporter used in a Q-PCR or RTQ-PCR reaction can be a labeledoligonucleotide probe. Probe based quantitative methods rely on thesequence-specific detection of amplification products of a desired DNAtemplate, using a labeled oligonucleotide. The oligonucleotide can be aprimer or a longer, different type of oligonucleotide. Theoligonucleotide can be DNA or RNA. As a result, unlike non-sequencespecific reporters, a labeled, sequence-specific probe hybridizes withseveral bases in an amplification product, and, thus, results inincreased specificity and sensitivity of detection. A label linked to aprobe can be any of the various reporters mentioned above and can alsoinclude a quencher (a molecule used, for example, to inhibitfluorescence). Methods for performing probe-based quantitativeamplification are described in U.S. Pat. No. 5,210,015, which isentirely incorporated herein by reference. Non-limiting examples ofprobes that can be useful in Q-PCR or RTQ-PCR reactions include TaqManprobes, TaqMan Tamara probes, TaqMan MGB probes, or Lion probes.

A variety of arrangements of quencher and fluorescent dye can be usedwhen both are used. In the case of a molecular beacon, for example, aquencher is linked to one end of an oligonucleotide capable of forming ahairpin structure. At the other end of the oligonucleotide is afluorescent dye. Unbound to a complementary sequence on an amplificationproduct, the oligonucleotide inter-hybridizes with itself and assumes ahairpin configuration. In the hairpin configuration, the fluorescent dyeand quencher are brought in close proximity which effectively preventsfluorescence of the dye. Upon hybridizing with an amplification productof a desired template DNA, however, the oligonucleotide hybridizes in alinear fashion, the fluorescence and quencher separate, and fluorescencefrom the dye can be achieved and subsequently detected. In otherexample, a linear, RNA based probe that includes a fluorescent dye and aquencher held in adjacent positions can be used for detection. The closeproximity of the dye to the quencher prevents its fluorescence. Upon thebreakdown of the probe with the exonuclease activity of a DNApolymerase, however, the quencher and dye are separated, and the freedye can fluoresce and be detected. As different probes can be designedfor different sequences, multiplexing is possible. In a multiplexeddetection, assaying for several DNA templates in the same reactionmixture can be possible by using different probes, each labeled with adifferent reporter, for each desired DNA template.

A Q-PCR or RTQ-PCR reaction can include a single reporter or can includemultiple reporters. One or more detection methodologies can be used forquantification. Moreover, as Q-PCR and RT-PCR generally adds just aquantification step, it can be generally linked to any type of PCRreaction.

LCR Reactions

The device, apparatus, system, and method of the current disclosure caninclude the completion of a LCR amplification reaction (or any step of aLCR reaction-as described elsewhere herein), and, thus, a sample cancomprise reagents necessary to complete a LCR amplification reaction.Non-limiting examples of such reagents include a template DNA moleculeto be amplified, a set of oligonucleotide probes that can each hybridizewith a different, but adjacent to the other, portion of a targetsequence on the template DNA, a DNA ligase, a buffer at a pH andconcentration suitable for a desired LCR reaction, a monovalent cation,and a divalent cation. Generally, the ratio of each reagent in thesample can vary and depend upon, for example, the amount of nucleic acidto be amplified and/or the desired amount of amplification products.Methods to determine the ratio of each reagent necessary for a LCRamplification reaction are generally known by those skilled in the art.

LCR is generally a method similar to PCR, with some important keydistinctions. A key distinction of general LCR over PCR, is that LCRamplifies an oligonucleotide probe using a DNA ligase enzyme to produceamplification products instead of through polymerization of nucleotideswith a DNA polymerase. In LCR, two complementary oligonucleotide probepairs that are specific to a DNA template can be used. Afterdenaturation of a to-be-replicated template DNA into its individualstrands, each probe pair can hybridize to adjacent positions on itsrespective individual strand of the template. Primers are generally notused in LCR. Any gap and/or nick created by the joining of two probescan be sealed by the enzyme DNA ligase, in order to produce a continuousstrand of DNA complementary to the template DNA. Similar to PCR, though,LCR generally requires thermal cycling, with each part of the thermalcycle driving a particular step of the reaction. Repeated temperaturechanges can result in the denaturation of the DNA template, annealing ofthe oligonucleotide probes, ligation of the oligonucleotide probes, andseparation of the ligated unit from the original DNA template. Moreover,a ligated unit synthesized in one thermal cycle can be replicated in thenext thermal cycle. Each thermal cycle can result in a doubling of theDNA template, resulting in exponential amplification of the template DNAin a fashion analogous to PCR.

Gap LCR Reactions

The device, apparatus, system, and method of the current disclosure caninclude the completion of a gap LCR amplification reaction, and, thus, asample can comprise reagents necessary to complete a gap LCRamplification reaction. Non-limiting examples of such reagents includethe reagents necessary to complete a LCR reaction, wherein the set ofoligonucleotide probes can each hybridize with a different, non-adjacentportion of a target sequence on the template DNA, dNTPs, and a DNApolymerase. Generally, the ratio of each reagent in the sample can varyand depend upon, for example, the amount of nucleic acid to be amplifiedand/or the desired amount of amplification products. Methods todetermine the ratio of each reagent necessary for a gap LCRamplification reaction are generally known by those skilled in the art.

Gap LCR is a specialized type of LCR that utilizes modifiedoligonucleotide probes that cannot be ligated if a specific sequence isnot present on a DNA template. The probes can be designed in a way thatwhen they hybridize to an individual strand of a DNA template, they doso discontinuously and are generally separated by a gap of one toseveral base pairs. The gap can be filled by with dNTPs using a DNApolymerase, which can result in adjacency of the two original probes. Asin general LCR, DNA ligase can join the two resulting, adjacent probesin order to produce a continuous strand of DNA complementary to theoriginal template. The newly synthesized strand can then be used forfurther thermal cycles of template amplification. Gap LCR generally hashigher sensitivity than LCR as it minimizes ligation where a desiredsequence is not present on a template DNA. Moreover, the combined use ofboth DNA ligase and DNA polymerase can also result in a more accurateidentification of a sequence of interest, even in cases where low levelsof DNA template are available.

Additionally, since LCR is a DNA replication method, analogous methodsto RT-PCR, Q-PCR, and RTQPCR are possible. For example, any of thereporters specified above can be considered for use in a quantitative(Q-LCR) or real-time quantitative LCR (RTQ-LCR) reaction. Moreover, LCRmethods can be combined with PCR or other nucleic amplificationtechniques.

Q-LCR and LTQ-LCR Reactions

The device, apparatus, system, and method of the current disclosure caninclude the completion of a Q-LCR or LTQ-PCR reaction, and, thus, asample can comprise reagents necessary to complete a Q-LCR or RTQ-LCRreaction. Non-limiting examples of such reagents include the reagentsnecessary to complete a LCR reaction and a reporter used to detectamplification products. Generally, the ratio of each reagent in thesample can vary and depend upon, for example, the amount of nucleic acidto be amplified and/or the desired amount of amplification products.Methods to determine the ratio of each reagent necessary for a Q-LCR andRTQ-LCR amplification reaction are generally known by those skilled inthe art.

Since LCR is a DNA replication method, analogous methods to RT-PCR,Q-PCR, and RTQPCR are possible. For example, any of the reportersspecified above can be considered for use in a quantitative (Q-LCR) orreal-time quantitative LCR (RTQ-LCR) reaction. Moreover, LCR methods canbe combined with PCR or other nucleic amplification techniques.

Digital Nucleic Acid Amplification Reactions

The device, apparatus, system, and method of the current disclosure caninclude the completion of a digital nucleic acid amplification reaction,and, thus, a sample can comprise reagents necessary to complete adigital nucleic acid amplification reaction. In general, any of theexample nucleic acid amplification reactions discussed herein can beconducted in digital form, upon proper separation of a sample and/orreagents necessary for nucleic acid amplification into smallerpartitions. In some embodiments, such partitions can be droplets or canbe larger aliquots of the original sample. Generally, the ratio of eachreagent in partitions can vary and depend upon, for example, the amountof nucleic acid to be amplified in each droplet and/or the desiredamount of amplification products. Methods to determine the ratio of eachreagent necessary for a particular digital nucleic acid amplificationreaction are generally known by those skilled in the art.

Digital nucleic acid amplification is a technique that allowsamplification of a subset of nucleic acid templates fractioned intopartitions obtained from a larger sample. In some cases, a partition cancomprise a single nucleic acid template, such that amplificationproducts generated from amplification of the template are exclusivelyderived from the template. Amplification products can be detected usinga reporter, including any of those example reporters described herein.The amplification of a single nucleic acid template can be useful indiscriminating genetic variations that include, for example, wild-typealleles, mutant alleles, maternal alleles, or paternal alleles of agene. More comprehensive discussions of this technology, with respect toPCR, can be found elsewhere-see Pohl et al., Expert Rev. Mo!. Diagn.,4(1):41-7 (2004), and Vogelstein and Kinzler, Proc. Natl. Acad. Sci. USA96:9236-9241 (1999), which are both incorporated herein in entirety byreference. So long as the proper thermal cycling of a partitioncomprising a complete reaction mixture (e.g., a reaction mixturecomprising both the nucleic acid template to be amplified and therequired reagents for the desired nucleic acid amplification reaction)is achieved, any of the example nucleic acid amplification reactionsdiscussed herein can be conducted digitally. Indeed, digital nucleicacid amplification methods still require thermal cycling and accuratetemperature control, as do their non-digital analogues.

In a digital nucleic acid amplification reaction, a large sample isfractioned into a number of smaller partitions, whereby the partitionscan contain on average a single copy of a nucleic acid template ormultiple copies of a template. Individual nucleic acid molecules can bepartitioned with the aid of a number of devices and strategies withnon-limiting examples that include micro-well plates, capillaries,dispersions that comprise emulsions, arrays of miniaturized chambers,nucleic acid binding surfaces, flow cells, droplet partitioning, orcombinations thereof. Each partition can be thermal cycled to generateamplification products of its component template nucleic acid, using anucleic acid amplification reaction of choice with non-limiting examplesof such reactions that include a digital PCR (dPCR) nucleic acidamplification reaction, a digital LCR (dLCR) nucleic acid amplificationreaction, a digital RT-PCR (dRT-PCR) nucleic acid amplificationreaction, a digital (dRT-LCR) nucleic acid amplification reaction, adigital Q-PCR (dQ-PCR) nucleic acid amplification reaction, a digitalQ-LCR (dQ-LCR) nucleic acid amplification reaction, a digital RTQ-PCR(dRTQ-PCR) nucleic acid amplification reaction, a digital LTQ-LCR(dLTQ-LCR) nucleic acid amplification reaction, or combinations thereof.

In cases where reporters are used, each partition can be considered“positive” or “negative” for a particular nucleic acid template ofinterest. The number of positives can be counted and, thus, one candeduce the starting amount of the template in the pre-partitioned samplebased upon the count. In some examples, counting can be achieved byassuming that the partitioning of the nucleic acid template populationin the original sample follows a Poisson distribution. Based on such ananalysis, each partition is labeled as either containing a nucleic acidtemplate of interest (e.g., labeled “positive”) or not containing thenucleic acid template of interest (e.g., labeled “negative”). Afternucleic acid amplification, templates can be quantified by counting thenumber of partitions that comprise “positive” reactions. Moreover,digital nucleic acid amplification is not dependent on the number ofamplification cycles to determine the initial amount of nucleic acidtemplate present in the original sample. This lack of dependencyeliminates relying on assumptions with respect to uncertain exponentialamplification, and, therefore, provides a method of direct, absolutequantification.

Most commonly, multiple serial dilutions of a starting sample are usedto arrive at the proper concentration of nucleic acid templates in thepartitions. The volume of each partition can depend on a host of factorsthat include, for example, the volume capacity of a thermal cycler usedto generate amplification products. Furthermore, quantitative analysesconducted by digital nucleic acid amplification can generally requirereliable amplification of single copies of nucleic acid template withlow false positive rates. Such capability can require carefuloptimization in microliter-scale vessels. Moreover, the analyticalprecision of a nucleic acid amplification reaction can be dependent onthe number of reactions.

In some embodiments, digital nucleic acid amplification reactions can bedroplet digital nucleic acid amplification reactions. Non-limitingexamples of such nucleic acid amplification reactions include dropletdigital PCR (ddPCR), droplet digital RT-PCR (ddRT-PCR), droplet digitalQ-PCR (ddQ-PCR), droplet digital RTQ-PCR (ddRTQ-PCR), droplet digitalLCR (ddLCR), droplet digital RT-LCR (ddRT-LCR), droplet digital Q-LCR(ddQ-LCR), or droplet digital RTQ-LCR (ddRTQ-PCR), or combinationsthereof.

In some cases, a digital nucleic acid amplification reaction can be adroplet digital nucleic acid amplification reaction. For example, such anucleic acid amplification reaction can be a droplet digital PCR (ddPCR)nucleic acid amplification reaction. A ddPCR nucleic acid amplificationreaction can be completed by first partitioning a larger samplecomprising nucleic acids into a plurality of droplets. Each dropletcomprises a random partition of nucleic acids in the original sample.The droplets can then be combined with different droplets that comprisethe reagents necessary for a PCR reaction (e.g., a set of two primersthat can hybridize with a target sequence on the template DNA, a DNApolymerase, deoxynucleotide triphosphates (dNTPs), a buffer at a pH andconcentration suitable for a desired PCR reaction, a monovalent cation,and a divalent cation). The new combined droplet is then properlythermal cycled in a thermal cycler and PCR commences. Alternatively, asample can already comprise reagents necessary for PCR prior topartitioning into droplets—droplet combination with other dropletswould, thus, not be required.

Analogous procedures can be followed to complete a droplet digitalRT-PCR (ddRT-PCR) nucleic acid amplification reaction, a droplet digitalLCR (ddLCR) nucleic acid amplification reaction, a droplet digitalRT-PCR (ddRT-LCR) nucleic acid amplification reaction, a droplet digitalQ-PCR (ddQ-PCR) nucleic acid amplification reaction, a droplet digitalRTQ-PCR (ddRTQ-PCR) nucleic acid amplification reaction, a dropletdigital Q-LCR (ddQ-LCR) nucleic acid amplification reaction, or adroplet digital RTQ-LCR (ddRTQ-LCR) reaction.

In the case of a quantitative droplet digital nucleic acid amplificationreaction (e.g., ddQ-PCR, ddRTQ-PCR, ddQ-LCR, or ddRTQ-LCR), droplets canalso comprise a reporter used to detect amplification products. Suchreporters can be contacted with nucleic acids by combining droplets orcan already be included in a partition comprising nucleic acid templatesto be amplified.

Droplet nucleic acid amplification can be completed using a variety ofsample holders. In some examples, droplets can be applied to one or morewells of a sample holder and then thermal cycled. In other examples, adevice comprising fluidic channels, such as, for example, a flow cell ormicrofluidic device can be used. Fluidic channels can be used totransport droplets through a sample holder (or other component of athermal cycler) such that droplet thermal contact with differenttemperature regions of the sample holder (or other component of athermal cycler) results in proper thermal cycling of the droplets.

Hinges, Opening Notches, Recessed Edge and Sliders

The devices, systems, and methods herein disclosed can include or useQ-cards for sample detection, analysis, and quantification. In someembodiments, the Q-card comprises hinges, notches, recesses, andsliders, which help to facilitate the manipulation of the Q card and themeasurement of the samples. The structure, material, function, variationand dimension of the hinges, notches, recesses, and sliders are hereindisclosed, or listed, described, and summarized in PCT Application(designating U.S.) Nos. PCT/US2016/046437 and PCT/US2016/051775, whichwere respectively filed on Aug. 10, 2016 and Sep. 14, 2016, U.S.Provisional Application No. 62/456,065, which was filed on Feb. 7, 2017,U.S. Provisional Application No. 62/456,287, which was filed on Feb. 8,2017, and U.S. Provisional Application No. 62/456,504, which was filedon Feb. 8, 2017, all of which applications are incorporated herein intheir entireties for all purposes.

Labels The devices, systems, and methods herein disclosed can employvarious types of labels that are used for analytes detection. The labelsare herein disclosed, or listed, described, and summarized in PCTApplication (designating U.S.) Nos. PCT/US2016/046437 andPCT/US2016/051775, which were respectively filed on Aug. 10, 2016 andSep. 14, 2016, U.S. Provisional Application No. 62/456,065, which wasfiled on Feb. 7, 2017, U.S. Provisional Application No. 62/456,287,which was filed on Feb. 8, 2017, and U.S. Provisional Application No.62/456,504, which was filed on Feb. 8, 2017, all of which applicationsare incorporated herein in their entireties for all purposes.

In some embodiments, labeling an analyte includes using, for example, alabeling agent, such as an analyte specific binding member that includesa detectable label. Detectable labels include, but are not limited to,fluorescent labels, colorimetric labels, chemiluminescent labels,enzyme-linked reagents, multicolor reagents, avidin-streptavidinassociated detection reagents, and the like. In some embodiments, thedetectable label is a fluorescent label. Fluorescent labels are labelingmoieties that are detectable by a fluorescence detector. For example,binding of a fluorescent label to an analyte of interest allows theanalyte of interest to be detected by a fluorescence detector. Examplesof fluorescent labels include, but are not limited to, fluorescentmolecules that fluoresce upon contact with a reagent, fluorescentmolecules that fluoresce when irradiated with electromagnetic radiation(e.g., UV, visible light, x-rays, etc.), and the like.

In some embodiments, suitable fluorescent molecules (fluorophores) forlabeling include, but are not limited to, IRDye800CW, Alexa 790, Dylight800, fluorescein, fluorescein isothiocyanate, succinimidyl esters ofcarboxyfluorescein, succinimidyl esters of fluorescein, 5-isomer offluorescein dichlorotriazine, cagedcarboxyfluorescein-alanine-carboxamide, Oregon Green 488, Oregon Green514; Lucifer Yellow, acridine Orange, rhodamine, tetramethylrhodamine,Texas Red, propidium iodide, JC-1(5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazoylcarbocyanineiodide), tetrabromorhodamine 123, rhodamine 6G, TMRM (tetramethylrhodamine methyl ester), TMRE (tetramethyl rhodamine ethyl ester),tetramethylrosamine, rhodamine B and 4-dimethylaminotetramethylrosamine,green fluorescent protein, blue-shifted green fluorescent protein,cyan-shifted green fluorescent protein, red-shifted green fluorescentprotein, yellow-shifted green fluorescent protein,4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid; acridine andderivatives, such as acridine, acridine isothiocyanate;5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS);4-amino-N-[3-vinylsulfonyl)phenyl]naphth-alimide-3,5 disulfonate;N-(4-anilino-1-naphthyl)maleimide; anthranilamide;4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a diaza-5-indacene-3-propionicacid BODIPY; cascade blue; Brilliant Yellow; coumarin and derivatives:coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin120),7-amino-4-trifluoromethylcoumarin (Coumarin 151); cyanine dyes;cyanosine; 4′,6-diaminidino-2-phenylindole (DAPI);5′,5″-dibromopyrogallolsulfonaphthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriaamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2-,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-(dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansylchloride);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives: eosin, eosin isothiocyanate, erythrosin and derivatives:erythrosin B, erythrosin, isothiocyanate; ethidium; fluorescein andderivatives: 5-carboxyfluorescein(FAM),5-(4,6-dichlorotriazin-2-yl)amino-fluorescein (DTAF),2′,7′dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), fluorescein,fluorescein isothiocyanate, QFITC, (XRITC); fluorescamine; IR144;IR1446; Malachite Green isothiocyanate; 4-methylumbelliferoneorthocresolphthalein; nitrotyrosine; pararosaniline; Phenol Red;B-phycoerythrin; ophthaldialdehyde; pyrene and derivatives: pyrene, 5pyrene butyrate, succinimidyl 1-pyrene; butyrate quantum dots; ReactiveRed 4 (Cibacron™ Brilliant Red 3B-A) rhodamine and derivatives:6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissaminerhodamine B sulfonyl chloride rhodamine (Rhod), rhodamine B, rhodamine123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101,sulfonyl chloride derivative of sulforhodamine 101 (Texas 10 Red);N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine;tetramethyl rhodamine isothiocyanate (TRITC); riboflavin;5-(2′-aminoethyl) aminonaphthalene-1-sulfonic acid (EDANS),4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL), rosolic acid; CALFluor Orange 560; terbium chelate derivatives; Cy 3; Cy 5; Cy 5.5; Cy 7;IRD 700; IRD 800; La Jolla Blue; phthalo cyanine; and naphthalo cyanine,coumarins and related dyes, xanthene dyes such as rhodols, resorufins,bimanes, acridines, isoindoles, dansyl dyes, aminophthalic hydrazidessuch as luminol, and isoluminol derivatives, aminophthalimides,aminonaphthalimides, aminobenzofurans, aminoquinolines,dicyanohydroquinones, fluorescent europium and terbium complexes;combinations thereof, and the like.

Suitable fluorescent proteins and chromogenic proteins include, but arenot limited to, a green fluorescent protein (GFP), including, but notlimited to, a GFP derived from Aequoria victoria or a derivativethereof, e.g., a “humanized” derivative such as Enhanced GFP; a GFP fromanother species such as Renilla reniformis, Renilla mulleri, orPtilosarcus guernyi; “humanized” recombinant GFP (hrGFP); any of avariety of fluorescent and colored proteins from Anthozoan species;combinations thereof; and the like.

In some embodiments, dyes that can be used to stain blood cells compriseWright's stain (Eosin, methylene blue), Giemsa stain (Eosin, methyleneblue, and Azure B), Can-Grünwald stain, Leishman's stain (“Polychromed”methylene blue (i.e. demethylated into various azures) and eosin),Erythrosine B stain (Erythrosin B), and other fluorescence stainincluding but not limit to Acridine orange dye,3,3-dihexyloxacarbocyanine (DiOC6), Propidium Iodide (PI), FluoresceinIsothiocyanate (FITC) and Basic Orange 21 (BO21) dye, Ethidium Bromide,Brilliant Sulfaflavine and a Stilbene Disulfonic Acid derivative,Erythrosine B or trypan blue, Hoechst 33342, Trihydrochloride,Trihydrate, and DAPI (4′,6-Diamidino-2-Phenylindole, Dihydrochloride).

Cloud. The devices, systems, and methods herein disclosed can employcloud technology for data transfer, storage, and/or analysis. Therelated cloud technologies are herein disclosed, or listed, described,and summarized in PCT Application (designating U.S.) Nos.PCT/US2016/046437 and PCT/US0216/051775, which were respectively filedon Aug. 10, 2016 and Sep. 14, 2016, U.S. Provisional Application No.62/456,065, which was filed on Feb. 7, 2017, U.S. ProvisionalApplication No. 62/456,287, which was filed on Feb. 8, 2017, and U.S.Provisional Application No. 62/456,504, which was filed on Feb. 8, 2017,all of which applications are incorporated herein in their entiretiesfor all purposes.

As used herein, the phrase, “for example,” the phrase, “as an example,”and/or simply the terms “example” and “exemplary” when used withreference to one or more components, features, details, structures,embodiments, and/or methods according to the present disclosure, areintended to convey that the described component, feature, detail,structure, embodiment, and/or method is an illustrative, non-exclusiveexample of components, features, details, structures, embodiments,and/or methods according to the present disclosure. Thus, the describedcomponent, feature, detail, structure, embodiment, and/or method is notintended to be limiting, required, or exclusive/exhaustive; and othercomponents, features, details, structures, embodiments, and/or methods,including structurally and/or functionally similar and/or equivalentcomponents, features, details, structures, embodiments, and/or methods,are also within the scope of the present disclosure.

As used herein, the phrases “at least one of” and “one or more of,” inreference to a list of more than one entity, means any one or more ofthe entity in the list of entity, and is not limited to at least one ofeach and every entity specifically listed within the list of entity. Forexample, “at least one of A and B” (or, equivalently, “at least one of Aor B,” or, equivalently, “at least one of A and/or B”) may refer to Aalone, B alone, or the combination of A and B.

As used herein, the term “and/or” placed between a first entity and asecond entity means one of (1) the first entity, (2) the second entity,and (3) the first entity and the second entity. Multiple entity listedwith “and/or” should be construed in the same manner, i.e., “one ormore” of the entity so conjoined. Other entity may optionally be presentother than the entity specifically identified by the “and/or” clause,whether related or unrelated to those entities specifically identified.

Where numerical ranges are mentioned herein, the invention includesembodiments in which the endpoints are included, embodiments in whichboth endpoints are excluded, and embodiments in which one endpoint isincluded and the other is excluded. It should be assumed that bothendpoints are included unless indicated otherwise. Furthermore, unlessotherwise indicated or otherwise evident from the context andunderstanding of one of ordinary skill in the art.

In the event that any patents, patent applications, or other referencesare incorporated by reference herein and (1) define a term in a mannerthat is inconsistent with and/or (2) are otherwise inconsistent with,either the non-incorporated portion of the present disclosure or any ofthe other incorporated references, the non-incorporated portion of thepresent disclosure shall control, and the term or incorporateddisclosure therein shall only control with respect to the reference inwhich the term is defined and/or the incorporated disclosure was presentoriginally.

II. Rapid Sample Temperature Changing for Assaying (VI)

The following disclosure is not intended to include all features andaspects of the present invention.

The present invention provides, among other things, the devices andmethods that can rapidly change or cycle (i.e. heating and cooling) asample temperature with high speed, less heating energy, high energyefficiency, a compact and simplified apparatus (e.g. handheld), easy andfast operation, and/or low cost.

The present invention has experimentally achieved a cycling of a sampletemperature between 95° C. and 55° C.) in a second or less.

The invention has six novel aspects (1) the devices and methods thatallow fast thermal cycling, (2) the devices and methods that allow thesample thickness uniform and sample holder mechanically stable forhandling, (3) simple operation, (3) devices and methods for doing realtime PCR (4) biochemistry, and (5) smartphone based systems.

To rapid thermal cycle the temperature of a sample or a portion of it,one must reduce the thermal mass and lateral heat.

Radiative heating and cooling are preferred.

A. Imager Based Rapid Temperature Assaying and Real Time PCR

1. Imaging Based Temperature Sensor

In certain embodiments, during a thermal cycling process, one or moretemperature sensing images are used to monitoring the temperature of asample. The temperature sensing image can sense a local temperature atdifferent locations of a sample. One can determine a suitable heatingtemperature to the sample (e.g. control the heating power), based on thetemperature map of a sample, rather than just a single lump-sumtemperature.

When heating up the assay device, some air bubbles and other defectswill form and be trapped in the heating area of the assay device. Andthe temperature of sample liquid and air are different. If using alump-sum temperature sensor to measure the average temperature in theheating area to be used as the temperature of sample liquid, it is notaccurate. In order to accurately measure the temperature of the sampleliquid in the heating area, an image-based temperature sensor should beused in the system to distinguish the temperature between air bubble andsample liquid.

FIG. 11A is the schematic of the system of heating and temperaturemonitoring device for the assay device. A heating source is put underthe assay device to heat up the assay device. And on top of the thermalimager, there is a thermal temperature sensor to monitor the temperatureof the heating area on the assay device. The temperature sensor is athermal imager whose field of view is aligned with the heating area.

FIG. 11B is the schematic of the field of view of the thermal imager inthe system described in FIG. 11A when heating up the assay device. Forexample, B1 and B2 are two air bubbles and/or defects which aregenerated and trapped in the assay device during heating up. And S3 isthe sample liquid region. Using the image based thermal sensor, we cantell the difference between the temperature of the sample liquid T3 andthe air bubble region temperatures T1 and T2. So that we can get moreaccurate temperature of the sample liquid in the heating area.

In this experiment, the assay device has a top (first) PMMA plate with50 um thickness, pillar array with 30 um pillar height, 30 um by 40 umpillar size, and 80 um inter pillar distance; a bottom (second) PETplate with 50 um thickness. A heating/cooling layer is on the outersurface of the second plate, and covers the entire second plate outersurface. The heating/cooling layer comprises an Au (gold) film and ablack paint layer. The gold film has one surface in contact with thesecond plate outer surface, and another surface being painted with ablack paint. The black paint is a commercial product of a filmcomposited of black carbon nanoparticle and polymer mixture. The blackpaint had an average thickness of ˜9 um (˜2 um thickness variation). Theblack paint layer may be directly facing incoming LED light. Between theAu film and the second plate outer surface, there is a 5 nm adhesionlayer of Ti, which improves the adhesion between Au and the secondplate. The heating source is a blue light emitting diode (LED) with acentral wavelength of 450 nm and power consumption around 500 mW.

-   -   9. In certain embodiments, a system, comprising:        -   (i) a device, comprising:        -   a first plate comprising a polymer material and having a            thickness less than or equal to 100 μm,        -   a second plate comprising a polymer material and having a            thickness less than or equal to 100 μm, wherein the second            plate is separated from the first plate in a parallel            arrangement by a distance less than or equal to the            thickness of the second plate,        -   a heating/cooling layer disposed on either the first plate            or the second plate, the heating/cooling layer having a            thickness and a thermal conductivity between 6×10⁻⁵ W/K            multiplied by the thickness of the heating/cooling layer and            1.5×10⁻⁴ W/K multiplied by the thickness of the            heating/cooling layer, and        -   a support frame configured to support at least one of the            first plate and the second plate;        -   an optical source configured to direct electromagnetic            radiation towards the heating/cooling layer,        -   a temperature sensor to monitor the temperature of the            heating area in the device;        -   wherein the heating/cooling layer is configured to absorb at            least a portion of the electromagnetic radiation such that            at least a portion of a liquid sample sandwiched between the            first plate and the second plate is heated at a rate of at            least 30° C./sec, and        -   wherein at least the portion of the liquid sample sandwiched            between the first plate and the second plate is cooled at a            rate of at least 30° C./sec when the heating/cooling layer            is not receiving the electromagnetic radiation generated by            the optical source, and        -   wherein the system consumes less than 500 mW of power.    -   10. The system of any prior embodiment, wherein the temperature        sensor is an image-based temperature sensor.    -   11. The system of any prior embodiment, wherein the temperature        sensor's field of view is 1 mm², 10 mm², 100 mm², 1000 mm², or        in a range between any of the two values.    -   12. The system of any prior embodiment, wherein the temperature        sensor's resolution is 1 um, 10 um, 100 um, 1 mm, or in a range        between any of the two values.    -   13. The system of any prior embodiment, wherein the working        thermal radiation wavelength of the temperature sensor's falls        in the range of 1 um to 10 um or 10 um to 100 um.    -   14. The system of any prior embodiment, wherein the thermal        sensor has a least a lens.    -   15. The system of any prior embodiment, wherein the thermal        sensor is an imager that can image at least a part of the        sample.    -   16. A method for measuring temperature of sample liquid in assay        device, comprising: imaging the heating area in the assay device        under thermal imager;        -   segmenting air bubble area or defect area and sample liquid            area in the image;        -   measuring the temperature of sample liquid area.            2. Real Time Detection (qPCR) Setup

In certain embodiments, a system comprise a assay device, the heater,and an optical monitor to monitor an optical signal from a sample in thecard, wherein the optical signal give an indication of a nucleic acidamplification inside the Q-card and the optical signal is monitoredduring a PCR process (that is a real time PCR).

In certain embodiments, the optical monitor is a photodetector. Incertain embodiments, the optical monitor is one or more imagers thatimage an area or a volume of the sample. Hence the image gives anoptical signal in each location of the sample being imaged. An analysisof the optical signal image can give more accurate analysis on thenucleic acid amplification than a lump-sum optical signal detection.

In certain embodiments, the nucleic amplification during a PCR processis monitored by an imager or more imagers, where the imagers image anarea or a volume of a sample and the signal of the imager represents thenucleic acid amplification by the PCR. In certain embodiments, signal isfluorescence signal. In certain embodiments, signal is a color signal.

The terms “assay device” and “sample holder” are interchangeable.

In the optical signal image analysis, in certain embodiments, artificialintelligence is used. In the optical signal image analysis, in certainembodiments, machine learning is used.

In certain embodiments, the system does real-time PCR by adding one ormulti fluorescent excitation light sources and detectors into theheating and temperature monitoring system.

In certain embodiments, a system has imagers for sample temperatureimaging and for nucleic acid amplification signals monitoring imaging.In certain embodiments, the sample temperature imaging and the nucleicacid amplification signal monitoring imaging uses a single opticalmonitor.

FIG. 12 is a schematic of the real-time PCR system comprising a heatingsource and temperature monitoring system as described FIG. 11 and a pairof fluorescent excitation light source and detector. In this case, theexcitation light source and fluorescence detector are on top of theassay device and aligned to the same excitation and detection area onthe assay device.

FIG. 13 is an example schematic of the real-time PCR system comprising aheating source, a fan and temperature detector as a temperature controlsystem and a pair of fluorescent excitation light source (with filter)and detector (with lens and filter) as the real time detection system;both temperature control system and real time detection system arecontrolled by Programmable logic controller (PLC). The PLC is furthercontrolled by an interface installed on a smartphone.

-   -   2. A system, comprising:        -   a device, comprising:        -   a first plate comprising a polymer material and having a            thickness less than or equal to 100 μm,        -   a second plate comprising a polymer material and having a            thickness less than or equal to 100 μm, wherein the second            plate is separated from the first plate in a parallel            arrangement by a distance less than or equal to the            thickness of the second plate,        -   a heating/cooling layer disposed on either the first plate            or the second plate, the heating/cooling layer having a            thickness and a thermal conductivity between 6×10⁻⁵ W/K            multiplied by the thickness of the heating/cooling layer and            1.5×10⁻⁴ W/K multiplied by the thickness of the            heating/cooling layer, and        -   a support frame configured to support at least one of the            first plate and the second plate;        -   an optical source configured to direct electromagnetic            radiation towards the heating/cooling layer,        -   a temperature sensor to monitor the temperature of the            heating area in the device;        -   a fluorescent excitation light source;        -   a fluorescent detector;        -   wherein the heating/cooling layer is configured to absorb at            least a portion of the electromagnetic radiation such that            at least a portion of a liquid sample sandwiched between the            first plate and the second plate is heated at a rate of at            least 30° C./sec, and        -   wherein at least the portion of the liquid sample sandwiched            between the first plate and the second plate is cooled at a            rate of at least 30° C./sec when the heating/cooling layer            is not receiving the electromagnetic radiation generated by            the optical source.    -   3. The system of any prior embodiment, wherein the excitation        light source can be but not limited to be a laser.    -   4. The system of any prior embodiment, wherein the excitation        light source can be but not limited to be a LED.    -   5. The system of any prior embodiment, wherein the fluorescent        detector is a photodetector.    -   6. The system of any prior embodiment, wherein the fluorescent        detector is mounted on an optical tube.    -   7. The system of any prior embodiment, wherein the fluorescent        detector is an image-based sensor.    -   8. A method for measuring fluorescence signal of sample liquid        in assay device, comprising:        -   imaging the heating area in the assay device under thermal            imager;        -   segmenting air bubble area or defect area and sample liquid            area in the image;        -   measuring the signal of sample liquid area.    -   9. A method for measuring fluorescence signal of sample liquid        in assay device, the time of measuring fluorescence signal is at        the primer annealing and extension of each cycle.    -   10. A method for measuring fluorescence signal of sample liquid        in assay device, the time of measuring fluorescence signal is at        the end of primer annealing and extension of each cycle.    -   11. A method for measuring fluorescence signal of sample liquid        in assay device, the time of measuring fluorescence signal is at        the time of heating source is off in each cycle.

In certain embodiments, an imager (either for temperature sensing or fornucleic acid amplification monitoring) in the present invention, isconnected to a computer, where various signal processing techniques,including machine learning is used. In certain embodiments, the signalprocessing results will be used to control the heating to the sample.

3. Heating Optical Pipe Structure

In certain embodiments, an optical pipe (also termed opticalcollimator), that collimates the light of a light source into theheating zone/plate, comprises a hollow tube with a reflective wall.

One embodiment of an optical pipe comprises a hollow structure (e.g.hollow tube) of round circle, rectangle, hexagonal, polygon, elliptic orcombination thereof.

One preferred embodiment of an optical pipe comprises a hexagonal hollowstructure.

One embodiment of an optical pipe comprises a hollow tube with areflective wall (i.e. its inner wall, outer wall, or both reflective).The reflective wall can be a thin light reflective coating on a wall ofthe hollow tube. The reflective coating can be a thin metal film, suchas gold, aluminum, silver, copper, or any mixture or combinationthereof.

In certain embodiments, the hollow structure is made of a dielectricmaterial of glasses, plastics, or a combination. In certain embodiments,the hollow structure is made of a metallic material.

FIG. 14 (a) shows a perspective view of a round heating tube and ahexagonal heating tube with a diameter of 6 mm and a point LED lightsource at the center of one tube end. (b) shows the optical beamintensity measured at the other end of tube. Clearly the hexagonalheating tube provides a more uniform distribution of heating lightintensity in the central 6 mm area.

In some embodiments, the hollow pipe has a length in the range of 1 mmto 70 mm, an inner dimension (diameter or width) in the range of 1 mm to40 mm, and a wall thickness in the range of 0.01 mm to 10 mm.

In some preferred embodiments, the hollow pipe for the light pipe has aninner diameter (or an average width) in a range of 1 mm to 5 mm, 5 mm to10 mm, 10 mm to 15 mm, 15 mm to 20 mm, 20 mm to 30 mm, or 30 mm to 50mm.

In some preferred embodiments, the hollow pipe for the light pipe has awall thickness (or an average width) in a range of 0.001 mm to 0.01 mm,0.01 mm to 0.1 mm, 0.1 mm to 0.5 mm, 0.5 mm to 1 mm, 1 mm to 2 mm, or 2mm to 50 mm.

Example. Fast SNAP PCR Amplification of PUC57 Plasmid DNA

The present technology uses the disclosed system for the PCRamplification of PUC57 plasmid DNA. The PCR reaction mixture wasprepared by mixing 10 uM PUC57 Forward primer, 10 uM PUC57 Reverseprimer and Cy5 labeled DNA probe with DNA buffer, 2.5 U/uL AptataqPolymerase, 25 mM MgCl2, dNTP, additives as Betaine, bovine serumalbumin (BSA), template DNA and ddH2O. 5 uL to 10 uL of the reaction wasadded onto the SNAP card and sealed for amplification.

In certain case, the whole card is incubated with 1% NaOH for 2 hoursunder 37° C., then washed with deionized water, then incubated with 4%bovine serum albumin (BSA) overnight under 4° C., washed with deionizedwater and dried at room temperature.

After amplification, the card is open and the production liquid issucked out for Gel electrophoresis analyze.

FIG. 15 shows a working SNAP PCR amplification of nucleic acid (E-coliplasmid DNA) with assay device demonstrating (a) 4.5 sec thermal cyclingtime (1 sec heating time from 60° C. to 95° C., 0.5 sec staying at 95°C., 2.5 sec cooling time from 95° C. to 60° C., and 0.5 sec staying at60° C.); (b) Gel electrophoresis results of SNAP PCR products ran in 3minutes (40 cycles) and conventional PCR products (40 cycles) ran in 40minutes shows 3 min SNAP PCR has a comparable amplification performanceas 40 min conventional PCR. The M line in the figure is a Gelelectrophoresis marker with 100 bp line marked. Both SNAP PCR andconventional PCR have clear 100 bp production line and similarintensity. Negative sample without template does not show bar in gelanalyze.

B. Clamping for Fast Thermal Change and/or Thermal Cycling

The present invention provides, among other things, devices and methodsto improve the time and energy needed in thermal cycling of a liquidsample by reducing the flow of the liquid sample from the inside to theoutside of a thermal cycling sample area.

In certain chemical, biological, or medical assays, a rapid change or arapid thermal cycling of a sample temperature is needed (e.g. Polymerasechain reaction (PCR) for amplifying nucleic acids).

During thermal cycling, a liquid sample will change in its volume withtemperature, and this can cause liquid sample flow. Liquid sample flowcan change the sample temperature and increase the time and energyneeded to do thermal cycling. Therefore, there is a need to reduce theliquid sample flow during thermal cycling.

One objective of the present invention is to address the need to reducethe liquid sample flow during thermal cycling. The present inventionadditionally provides devices and methods for isothermal nucleic acidamplification.

According to the present invention, a sample holder comprised a firstplate and a second plate, where a liquid sample is sandwiched betweenthe plates. In some embodiments, the two plate are fixed to each other.In some embodiments, the two plates are movable relative to each other.In some embodiments, there are spacers between the two plates toregulate the spacing between the two plates.

According to the present invention, a clamp structure comprises tworings, wherein the clamp has different configuration: inactiveconfiguration and active configuration. In an inactive configuration,the two rings of the claim are open and the two rings do not insert anycompression force on the sample plates. And in an activationconfiguration, the rings are being pushed towards to each other andinsert a compressing force on the areas of a sample holder that areunder the rings, and the comprising pinch force pinches the sampleholder area under the ring together. In some embodiments, the pinchingof the sample holder can reduce the sample in the inside of the clampring to flow to the outside of the clamp.

According the present invention, during a thermal cycling, a clamp isused and active, and the use of the clamp reduces the flow of the liquidsample in the inside of the ring to the outside of the ring. The use ofthe claim reduces the liquid sample flow and hence the energy exchangebetween the sample in the inside of the ring to the outside of the ring,and reduce the heating energy for heating up the sample inside of theclamp, and increase the thermal cycling time.

According the present invention, during a thermal cycling, the use of aclamp can reduce air bubbles during thermal cycling, which in turnimprove thermal cycling quality.

FIG. 16 shows a sectional view of an embodiment of the system of thepresent invention, comprising a sample holder and a clamping structure.The active clamp. Panel (A) illustrates the system before the clampingstructure is activated, where the two rings of the clamp are open andhence does not assert a force to push the two plate together. Panel (B)illustrates that the clamp is activated, where a force is applied by theclamping structure to pinch the sample holder area that is pressed bythe claim. The activation of the clamping structure, which clamps thearea of the sample holder under the claim, will prevent a fluid samplefrom flowing outside the chamber as heat is delivered theheating/cooling layer. In some embodiments, the first plate and thesecond plate are fixed relative to each other. In some embodiments, thefirst plate and the second plate are movable relative to each other.

FIG. 17 shows a top view of an embodiment of one side of a ring clamp.Each ring has a width and a circumference. It shows a circular shapedring clamp and a rectangle shaped ring clamp. In some embodiments, thering clamp has other shapes including, but not limited to,

round circle, rectangle, hexagonal, polygon, elliptic or combinationthereof.

FIG. 18 shows a sectional view of an embodiment of the system of thepresent invention, comprising a sample holder, and a clamping structure.The sample holder comprises a first plate and a second plate that aremovable to each other, where in the second plate a well. Panel (A)illustrates the system before the ring clamping structure is activated,where the two rings of the clamp does not assert a force to push the twoplate together. Panel (B) illustrates the system after a force isapplied by the clamping structure. The activation of the clampingstructure will prevent a fluid sample from flowing outside the chamberas heat is delivered the heating/cooling layer.

FIG. 19 . shows a sectional view of an embodiment of the system of thepresent invention, comprising a sample holder and a clamping structure.The sample holder comprises a first plate, a second plate with spacersthat are fixed on the inner surface. Panel (A) illustrates the systembefore the clamping structure is activated. Panel (B) illustrates thesystem after a force is applied by the clamping structure. Theactivation of the clamping structure will prevent a fluid sample fromflowing outside the chamber as heat is delivered the heating/coolinglayer.

FIG. 20 . shows a sectional view of an embodiment of the system of thepresent invention, comprising a first plate, a second plate with a welland spacers that are fixed on the inner surface, and a clampingstructure. Panel (A) illustrates the system before the clampingstructure is activated. Panel (B) illustrates the system after a forceis applied by the clamping structure. The activation of the clampingstructure will prevent a fluid sample from flowing outside the chamberas heat is delivered the heating/cooling layer.

FIG. 21 . shows exemplary embodiments of two types of clampingstructures. Panel (A) comprises a support with a one-spring ringstructure. Panel (B) comprises a support with a four-spring ringstructure.

In one aspect, the present invention provides a device for rapidlychanging the temperature of a fluidic sample, comprising: a first plate,a second plate, and a clamping structure, wherein:

-   -   iii. the first plate and the second plate have on their inner        surface a sample contact area for contacting a fluidic sample;        wherein the sample contact area of the first plate and the        second plate face each other, are separated by a distance of 200        um or less, and are capable of sandwiching the sample between        them;    -   iv. the clamping structure comprises a top ring and a bottom        ring that are movable to each other and comprise:        -   a. an open configuration, wherein the top ring and the            bottom ring do not push the first plate and second plate            together; and        -   b. a closed configuration, wherein the top ring and the            bottom ring assert a force again each other to push the            first plate and the second plate in the closed            configuration, so the flow of a sample from the inside to            the outside of a ring area during thermal cycling is reduced            compared to without using a clamping structure.

In one aspect, the present invention provides a device for rapidlychanging the temperature of a fluidic sample, comprises: a first plate,a second plate with a well, and a clamping structure, wherein:

-   -   iii. the first plate and the second plate with a well have on        their inner surface a sample contact area for contacting a        fluidic sample; wherein the sample contact area of the first        plate and the second plate face each other, are separated by a        distance of 200 um or less, and are capable of sandwiching the        sample between them;    -   iv. the clamping structure comprises a top ring and a bottom        ring that are movable to each other and comprise:        -   c. an open configuration, wherein the top ring and the            bottom ring do not push the first plate and the second plate            with a well together; and        -   d. a closed configuration, wherein the top ring and the            bottom ring assert a force again each other to push the            first plate and the second plate with a well in the closed            configuration, so the flow of a sample from the inside to            the outside of a ring area during thermal cycling is reduced            compared to without using a clamping structure.

As illustrated in FIG. 17 (Panel A and Panel B) and FIG. 18 (Panel A andPanel B), the sectional views of certain embodiments show an openconfiguration (before clamp activation) and a closed configuration(after the clamp is activated).

In certain embodiments, the first plate and second plate are flat. FIG.17 illustrates an exemplary flat plate.

In certain embodiments, the second plate further comprises a wall. FIG.18 illustrates an exemplary well in the second plate.

In one aspect, the present invention provides a device for rapidlychanging the temperature of a fluidic sample, comprises: a first plate,a second plate, spacers, and a clamping structure, wherein:

-   -   iv. the first plate and/or second plate comprise spacers fixed        to the inner surface of the first plate and/or second plate,    -   v. the first plate and the second plate comprise on their inner        surface a sample contact area for contacting a fluidic sample;        wherein the sample contact area of the first plate and the        second plate face each other, are separated by a distance of 200        um or less, and are capable of sandwiching the sample between        them,    -   vi. the clamping structure comprises a top ring and a bottom        ring that are movable to each other and comprise:        -   c. an open configuration, wherein the top ring and the            bottom ring do not push the first plate and the second plate            with a well together; and        -   d. a closed configuration, wherein the top ring and the            bottom ring assert a force again each other to push the            first plate and the second plate with a well in the closed            configuration, so the flow of a sample from the inside to            the outside of a ring area during thermal cycling is reduced            compared to without using a clamping structure.

In one aspect, the present invention provides a device for rapidlychanging the temperature of a fluidic sample, comprises: a first plate,a second plate with a well, spacers, and a clamping structure, wherein:

-   -   iv. the first plate and/or second plate with a well comprise        spacers fixed to the inner surface of the first plate and/or        second plate,    -   v. the first plate and the second plate with a well comprise on        their inner surface a sample contact area for contacting a        fluidic sample; wherein the sample contact area of the first        plate and the second plate face each other, are separated by a        distance of 200 um or less, and are capable of sandwiching the        sample between them,    -   vi. the clamping structure comprises a top ring and a bottom        ring that are movable to each other and comprise:        -   c. an open configuration, wherein the top ring and the            bottom ring do not push the first plate and the second plate            with a well together; and        -   d. a closed configuration, wherein the top ring and the            bottom ring assert a force again each other to push the            first plate and the second plate with a well in the closed            configuration, so the flow of a sample from the inside to            the outside of a ring area during thermal cycling is reduced            compared to without using a clamping structure.

As illustrated in FIG. 19 (Panel A and Panel B) and FIG. 20 (Panel A andPanel B), the sectional views of the embodiments show an openconfiguration (before clamp activation) and a closed configuration(after the clamp is activated) wherein spacers are positioned betweenthe first plate and the second plate to regulate the distance betweenthe two plates (i.e., the spacing of the first plate and the secondplate), to regulate the sample thickness. The spacers allow thethickness of the sample between the first plate and the second plate tobe uniform over a large area, even when the first plate and second plateare thin and flexible.

FIG. 19 illustrates certain embodiments where the first plate and thesecond plate are flat.

FIG. 20 illustrates certain embodiments where the second plate furthercomprises a well.

In one aspect, the present invention provides a device for rapidlychanging the temperature of a fluidic sample, comprising: a first plate,a second plate, and a clamping structure, wherein:

-   -   iii. the first plate and the second plate have on their inner        surface a sample contact area for contacting a fluidic sample;        wherein the sample contact area of the first plate and the        second plate face each other, are separated by a distance of 200        um or less, and are capable of sandwiching the sample between        them;    -   iv. the clamping structure comprises a top ring and a bottom        ring that are movable to each other and comprise:        -   a. an open configuration, wherein the top ring and the            bottom ring do not push the first plate and second plate            together; and        -   b. a closed configuration, wherein the top ring and the            bottom ring assert a force again each other to push the            first plate and the second plate in the closed            configuration, so the flow of a sample from the inside to            the outside of a ring area during thermal cycling is reduced            compared to without using a clamping structure.

In one aspect, the present invention provides a device for rapidlychanging the temperature of a fluidic sample, comprising: a first plate,a second plate with a well, and a clamping structure, wherein:

-   -   v. the first plate and the second plate with a well have on        their inner surface a sample contact area for contacting a        fluidic sample; wherein the sample contact area of the first        plate and the second plate face each other, are separated by a        distance of 200 um or less, and are capable of sandwiching the        sample between them;    -   vi. the clamping structure comprises a top ring and a bottom        ring that are movable to each other and comprise:        -   d. an open configuration, wherein the top ring and the            bottom ring do not push the first plate and the second plate            with a well together; and        -   e. a closed configuration, wherein the top ring and the            bottom ring assert a force again each other to push the            first plate and the second plate with a well in the closed            configuration, so the flow of a sample from the inside to            the outside of a ring area during thermal cycling is reduced            compared to without using a clamping structure.

In one aspect, the present invention provides a device for rapidlychanging the temperature of a fluidic sample, comprising: a first plate,a second plate, spacers, and a clamping structure, wherein:

-   -   iv. the first plate and/or second plate comprise spacers fixed        to the inner surface of the first plate and/or second plate,    -   v. the first plate and the second plate comprise on their inner        surface a sample contact area for contacting a fluidic sample;        wherein the sample contact area of the first plate and the        second plate face each other, are separated by a distance of 200        um or less, and are capable of sandwiching the sample between        them,    -   vi. the clamping structure comprises a top ring and a bottom        ring that are movable to each other and comprise:        -   c. an open configuration, wherein the top ring and the            bottom ring do not push the first plate and the second plate            with a well together; and        -   d. a closed configuration, wherein the top ring and the            bottom ring assert a force again each other to push the            first plate and the second plate with a well in the closed            configuration, so the flow of a sample from the inside to            the outside of a ring area during thermal cycling is reduced            compared to without using a clamping structure.

In one aspect, the present invention provides a device for rapidlychanging the temperature of a fluidic sample, comprising: a first plate,a second plate with a well, spacers, and a clamping structure, wherein:

-   -   iv. the first plate and/or second plate with a well comprise        spacers fixed to the inner surface of the first plate and/or        second plate,    -   v. the first plate and the second plate with a well comprise on        their inner surface a sample contact area for contacting a        fluidic sample; wherein the sample contact area of the first        plate and the second plate face each other, are separated by a        distance of 200 um or less, and are capable of sandwiching the        sample between them,    -   vi. the clamping structure comprises a top ring and a bottom        ring that are movable to each other and comprise:        -   c. an open configuration, wherein the top ring and the            bottom ring do not push the first plate and the second plate            with a well together; and        -   d. a closed configuration, wherein the top ring and the            bottom ring assert a force again each other to push the            first plate and the second plate with a well in the closed            configuration, so the flow of a sample from the inside to            the outside of a ring area during thermal cycling is reduced            compared to without using a clamping structure.

In one aspect, the present invention provides a method for rapidlychanging the temperature of a fluidic sample, comprising: a first plate,a second plate, and a clamping structure, wherein:

-   -   iii. the first plate and the second plate have on their inner        surface a sample contact area for contacting a fluidic sample;        wherein the sample contact area of the first plate and the        second plate face each other, are separated by a distance of 200        urn or less, and are capable of sandwiching the sample between        them;    -   iv. the clamping structure comprises a top ring and a bottom        ring that are movable to each other and comprise:        -   a. an open configuration, wherein the top ring and the            bottom ring do not push the first plate and second plate            together; and        -   b. a closed configuration, wherein the top ring and the            bottom ring assert a force again each other to push the            first plate and the second plate in the closed            configuration, so the flow of a sample from the inside to            the outside of a ring area during thermal cycling is reduced            compared to without using a clamping structure.

In one aspect, the present invention provides a method for rapidlychanging the temperature of a fluidic sample, comprising: a first plate,a second plate with a well, and a clamping structure, wherein:

-   -   i. the first plate and the second plate with a well have on        their inner surface a sample contact area for contacting a        fluidic sample; wherein the sample contact area of the first        plate and the second plate face each other, are separated by a        distance of 200 um or less, and are capable of sandwiching the        sample between them;    -   ii. the clamping structure comprises a top ring and a bottom        ring that are movable to each other and comprise:        -   a) an open configuration, wherein the top ring and the            bottom ring do not push the first plate and the second plate            with a well together; and        -   b) a closed configuration, wherein the top ring and the            bottom ring assert a force again each other to push the            first plate and the second plate with a well in the closed            configuration, so the flow of a sample from the inside to            the outside of a ring area during thermal cycling is reduced            compared to without using a clamping structure.

In one aspect, the present invention provides a method for rapidlychanging the temperature of a fluidic sample, comprising: a first plate,a second plate, spacers, and a clamping structure, wherein:

-   -   iv. the first plate and/or second plate comprise spacers fixed        to the inner surface of the first plate and/or second plate,    -   v. the first plate and the second plate comprise on their inner        surface a sample contact area for contacting a fluidic sample;        wherein the sample contact area of the first plate and the        second plate face each other, are separated by a distance of 200        um or less, and are capable of sandwiching the sample between        them,    -   vi. the clamping structure comprises a top ring and a bottom        ring that are movable to each other and comprise:        -   c. an open configuration, wherein the top ring and the            bottom ring do not push the first plate and the second plate            with a well together; and        -   d. a closed configuration, wherein the top ring and the            bottom ring assert a force again each other to push the            first plate and the second plate with a well in the closed            configuration, so the flow of a sample from the inside to            the outside of a ring area during thermal cycling is reduced            compared to without using a clamping structure.

In one aspect, the present invention provides a method for rapidlychanging the temperature of a fluidic sample, comprising: a first plate,a second plate with a well, spacers, and a clamping structure, wherein:

-   -   iv. the first plate and/or second plate with a well comprise        spacers fixed to the inner surface of the first plate and/or        second plate,    -   v. the first plate and the second plate with a well comprise on        their inner surface a sample contact area for contacting a        fluidic sample; wherein the sample contact area of the first        plate and the second plate face each other, are separated by a        distance of 200 um or less, and are capable of sandwiching the        sample between them,    -   vi. the clamping structure comprises a top ring and a bottom        ring that are movable to each other and comprise:        -   c. an open configuration, wherein the top ring and the            bottom ring do not push the first plate and the second plate            with a well together; and        -   d. a closed configuration, wherein the top ring and the            bottom ring assert a force again each other to push the            first plate and the second plate with a well in the closed            configuration, so the flow of a sample from the inside to            the outside of a ring area during thermal cycling is reduced            compared to without using a clamping structure.

In certain embodiments, the device further comprises a heating layer.

In certain embodiments, the heating layer is positioned on the innersurface, the outer surface, or on the inside of one of the plates.

In certain embodiments, the heating layer is configured to heat arelevant volume of the sample, wherein the relevant volume of the sampleis a portion or an entirety of the sample that is being heated to adesired temperature.

In certain embodiments, the device further comprising a cooling layer.

In certain embodiments, the cooling layer is positioned on the innersurface, the outer surface, or inside one of the plates.

In certain embodiments, the cooling layer is configured to cool therelevant sample volume.

In certain embodiments, the cooling layer comprises a layer of materialthat has a thermal conductivity to thermal capacity ratio of 0.6 cm²/secor larger.

In certain embodiments, the clamping structure is attached to either oneor both of the first and second plates, and wherein the clampingstructure is configured to hold the device and regulate the thickness ofthe sample layer during the heating of the device.

In certain embodiments, the clamp is in a shape such as, but not limitedto, circle, triangle, round, elliptical, polygon, or any superpositionof these shapes.

In certain preferred embodiments, the shape of the clamp is circle andelliptical.

In certain embodiments, the width of the clamp is 100 um, 300 um, 500um, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 10 mm, or in a range between any ofthe two values.

In certain preferred embodiments, the width of the clamp is 1 mm, 2 mm,and 3 mm.

In certain embodiments, the thickness of the clamp is 100 um, 300 um,500 um, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 10 mm, 15 mm, 20 mm, 30 mm or in arange between any of the two values.

In certain preferred embodiments, the thickness of the clamp is 1 mm, 2mm, 10 mm, and 15 mm.

In certain embodiments, the circumference of the clamp is 5 mm, 10 mm,20 mm, 30 mm, 38 mm, 50 mm, 62 mm, 100 mm or in a range between any ofthe two values.

In certain preferred embodiments, the circumference of the clamp is 38mm and 62 mm.

In certain preferred embodiments, the pressure provided by clamp on thecard is 5 PSI, 10 PSI, 30 PSI, 60 PSI, 90 PSI, 100 PSI, 150 PSI, 200PSI, 500 PSI or in a range between any of the two values.

In certain preferred embodiments, the pressure provided by clamp on thecard is less than 30 PSI, less than 60 PSI, and less than 90 PSI.

In certain embodiments, the material of clamp includes, but not limitedto, glass, quartz, oxides, silicon-dioxide, silicon-nitride, hafniumoxide (HfO), aluminum oxide (AlO), semiconductors: (silicon, GaAs, GaN,etc.).

In certain preferred embodiments, the material of clamp includes, butnot limited to metals (e.g. gold, silver, coper, aluminum, Ti, Ni,etc.), ceramics, or any combinations of thereof.

In certain preferred embodiments, the material of clamp includes, butnot limited to, polymers (e.g. plastics) or amorphous organic materials.The polymer materials include, but not limited to, acrylate polymers,vinyl polymers, olefin polymers, cellulosic polymers, noncellulosicpolymers, polyester polymers, Nylon, cyclic olefin copolymer (COC),poly(methyl methacrylate) (PMMA), polycarbonate (PC), cyclic olefinpolymer (COP), liquid crystalline polymer (LCP), polyimide (PA),polyethylene (PE), polyimide (PI), polypropylene (PP), poly(phenyleneether) (PPE), polystyrene (PS), polyoxymethylene (POM), polyether etherketone (PEEK), polyether sulfone (PES), poly(ethylene phthalate) (PET),polytetrafluoroethylene (PTFE), polyvinyl chloride (PVC), polyvinylidenefluoride (PVDF), polybutylene terephthalate (PBT), fluorinated ethylenepropylene (FEP), perfluoroalkoxyalkane (PFA), polydimethylsiloxane(PDMS), rubbers, or any combinations of thereof.

In certain preferred embodiments, the material of clamp includes, butnot limited to glass, quartz, oxides, silicon-dioxide, silicon-nitride,hafnium oxide (HfO), aluminum oxide (AlO), semiconductors: (silicon,GaAs, GaN, etc.), plastics, metals (e.g. gold, silver, coper, aluminum,Ti, Ni, etc.), ceramics, or any combinations of thereof.

C. Fast Temperature Changes

Working Principle

One aspect of the present invention is to reduce thermal cycling time,to reduce the heating energy used for such cycling, to increase energyefficiency, and to reduce total power consumption.

The thermal cycling time (speed), heating energy, energy efficiency, andpower consumption are related. When more heating energy is needed inraising the temperature of a given sample, the more energy must beremoved in cooling the sample, which, in turn, needs more time and/ormore energy to perform the cooling.

Many thermal cyclers in prior art require a use of a significant amountof heating energy to the sample holder (e.g. plastic chamber walls)rather than to the sample; a use of lateral thermal conduction throughlarge thermal mass and poor-thermal conduction materials of the sampleholder as the major cooling channel to cool the sample (note that amaterial needs to absorb and release energy to perform a thermalconduction); a use of conductive cooling as major cooling method, and/ora use of an extra cooling gas or a moving cooling block. Theseapproaches lead to issues of long thermal cycling time, high heatingenergy, low energy efficiency, bulky apparatus, and/or high cost.

Based on theoretical and experimental investigations, the presentinvention provides solutions to certain drawbacks in a sample thermalcycling in the prior arts.

To illustrate the working principle of the present invention, let uslook at the energy components in heating and cooling a sample by athermal cycler. The heating and cooling share three energy components:(i) one related to thermal mass (i.e. a material's ability to absorb andstore energy; larger the thermal mass, more energy needed to be addedfor heating up and more energy needed to be removed in cooling), (ii)heat loss by thermal radiation, and (iii) heat loss by thermalconduction/convection. To heat fast, all three energy components need tobe small. But to cool fast, the first energy component needs to besmall, but at least one of the last two energy components needs to belarge.

Through theoretical and experimental investigation, the presentinvention balances and/or optimizes the three energy components forachieving rapid heating and cooling. Particularly, in certainembodiments, the present invention reduces the thermal mass that must beheated in a thermal cycle, limits lateral thermal conduction, and usesradiative heat loss as a primary way to remove energy from the heatedsample.

According to the present invention, the cooling of a sample issignificantly by thermal radiative cooling, not by thermal conductioncooling. Therefore, in a thermal cycling, most or a significant part ofthe non-sample materials on a sample holder do not absorb and release asmuch energy as that in a thermal conduction dominated system.

One aspect of the present invention provides devices and methods thatreduce the heating to non-sample materials on the sample holder.

Another aspect of the present invention provides devices and methodsthat reduce lateral thermal conduction through large thermal mass andpoor-thermal conduction materials on the sample holder.

Another aspect of the present invention provides devices and methodsthat use thermal radiative cooling as the major cooling channel to coolthe sample.

Another aspect of the present invention provides devices and methodsthat place spacers between to plates (i.e. walls) that sandwich asample. The spacers provides good sample uniformity over a large area,even when the plates are thin (e.g. 25 um thick) and flexible. Withoutspacers, it can be difficult to achieve a uniform sample thickness, whenthe two plates that confine the sample become very thin.

Another aspect of the present invention provides devices and methodsthat make the device operation easier.

According to the present invention, the thermal radiative cooling uses amaterial layer are configured (in terms of materials and shape) that hasgood thermal radiative cool properties during the cooling, and a lowthermal mass (hence a low heating energy) during heating.

According to the present invention, the sample holder is configured tolimit/minimize the thermal conduction cooling.

According to the present invention, the sample thickness, the firstplate and the second plate (which are facing each other) of the samplechamber wall thickness are configured to reduce the lateral thermalconduction (i.e. in the direction of the plate).

According to the present invention, in some embodiments, the thermalradiative cooling layer is the same heating/cooling layer of the heatinglayer, but the ratio of the cooling zone to the heating zone, thematerial properties, and the material thickness and geometry areconfigured to make the heating/cooling layer has a low thermal mass inheating and high rate of thermal radiative cooling.

Another objective of the present invention is to make one cycle of asample temperature change (e.g. from 95° C. to 55° C.) in a few secondsor even sub-second (e.g., 0.7 second).

Another aspect of the present invention is that it provides usefuldevices and methods for isothermal nucleic acid amplification, where asample temperature needs to be raised from environment to an elevatedtemperature (i.e. 65° C.) and keep there for a period of time (i.e.,5-10 min). One aspect of the present invention is to raise thetemperature fast, to use less energy, and to make the apparatus compact,lightweight, and portable.

One aspect of the present invention is that the thermal masses of thecard as well as the sample are minimized to reduce the energy needed forheating and the energy to be removed for cooling.

Another aspect of the present invention is that in certain embodiments,only a small portion of the sample is heated and/or cooled.

Another aspect of the present invention is that it uses a thin highthermal conductivity layer that has an area size larger than that of therelevant sample area.

Another aspect of the present invention is that it uses a thin highthermal conductivity layer that has an area size larger than the heatingzone area.

Another aspect of the present invention provides devices and methodsthat reduce the heating to non-sample materials on the sample holder.

Another aspect of the present invention provides devices and methodsthat reduce lateral thermal conduction in large thermal mass andpoor-thermal conduction materials on the sample holder.

Another aspect of the present invention provides devices and methodsthat use thermal radiative cooling as the major cooling channel to coolthe sample.

Another aspect of the present invention is that it can achieve fastthermal cycling without using a cooling gas.

Another aspect of the present invention is that the thermal masses ofthe card as well as the sample are minimized to reduce the energy neededfor heating and the energy to be removed for cooling.

Another aspect of the present invention is that the radiative coolingand convention cooling are adjusted for rapid cooling.

Another aspect of the present invention is that heat sink for radiativecooling and/or convention cooling is used for rapid cooling.

One embodiment of a sample thermal cycling apparatus in the presentinvention (as illustrated in FIG. 22 ) comprises: (i) a sample holder,termed “RHC (rapid heating and cooling) Card” or “sample card”, thatallows a rapid heating and cooling of a sample on the card; (ii) aheating source, (iii) an extra heat sink (optional), (iv) a temperaturecontrol system, and (v) a signal monitoring system (optional). Thetemperature control system and signal monitoring system are notexplicitly illustrated in FIG. 22 , but may be used to control theoutput of the heating source. In some embodiments, a signal sensor isincluded to detect optical signals from samples on the sample holder.Note that certain embodiments of the present invention can have just oneor several components illustrated in FIG. 22 .

FIGS. 23A and 23B show sectional views of two embodiments of the deviceof the present invention. FIG. 23A shows an embodiment comprising aseparate heating layer (112-1) and a separate cooling layer (112-2),wherein the heating layer (112-1) is on the outer surface of one of theplates and the cooling layer (112-2) is on the outer surface of theother plate. FIG. 23B shows an embodiment comprising a heating layer(112-1) and a cooling layer (112-2), wherein the heating layer (112-1)and the cooling layer (112-2) are structurally distinct but in contactwith each other, and the two layers are both on the outer surface of oneof the plates.

SH-1 One detailed description of one embodiment of a RHC card in thepresent invention is that a device for rapidly changing the temperatureof a fluidic sample, comprising:

a first plate (10), a second plate (20), a heating layer (112-1), and acooling layer (112-2), wherein:

each of the first plate and the second plate has, on its respectiveinner surface, a sample contact area for contacting a fluidic sample;wherein the sample contact areas face each other, are separated by anaverage separation distance of 200 um or less, and are capable ofsandwiching the sample between them;

the heating layer is:

positioned on the inner surface, the outer surface, or inside of one ofthe plates, and

configured to heat a relevant volume of the sample, wherein the relevantvolume of the sample is a portion or an entirety of the sample that isbeing heated to a desired temperature; and

the cooling layer is:

positioned on the inner surface, the outer surface, or inside of one ofthe plates, and

configured to cool the relevant sample volume; and

comprises a layer of material that that has a thermal conductivity tothermal capacity ratio of 0.6 cm²/sec or larger;

wherein the distance between the cooling layer and a surface of therelevant sample volume is zero or less than a distance that isconfigured to make the thermal conductance per unit area between thecooling layer and the surface of the relevant sample volume equal to 70W/(m²·K) or larger; and

wherein, in some embodiments, the heating layer and cooling layer arethe same material layer that have a heating zone and cooling zone, andwherein the heating zone and cooling zone can have the same area ordifferent areas.

SH-2 Another detailed description of one embodiment of a RHC card(sample holder) in the present invention is that a device for rapidlychanging the temperature of a fluidic sample, comprising:

A first plate (10), a second plate (20), a heating layer (112-1), and acooling layer (112-2), wherein:

each of the first plate and the second plate has, on its respectiveinner surface, a sample contact area for contacting a fluidic sample;wherein the sample contact areas face each other, are separated by anaverage separation distance of 200 um or less, and are capable ofsandwiching the sample between them;

the heating layer is:

positioned on the inner surface, the outer surface, or inside of one ofthe plates, and

configured to heat a relevant volume of the sample, wherein the relevantvolume of the sample is a portion or an entirety of the sample that isbeing heated to a desired temperature; and

the cooling layer is:

positioned on the inner surface, the outer surface, or inside of one ofthe plates; and

configured to cool the relevant sample volume; and

comprises a layer of material that that has a thermal conductivity tothermal capacity ratio of 0.6 cm²/sec or larger, wherein the highthermal conductivity to thermal capacity ratio layer has an area largerthan the lateral area of the sample volume;

wherein the distance between the cooling layer and a surface of therelevant sample volume is zero or less than a distance that isconfigured to make the thermal conductance per unit area between thecooling layer and the surface of the relevant sample volume equal to 150W/(m²·K) or larger; and

wherein, in some embodiments, the heating layer and cooling layer arethe same material layer that have a heating zone and cooling zone, andwherein the heating zone and cooling can have the same area or differentareas.

As illustrated in FIGS. 24A and 24B, in some embodiments of the presentinvention, the heating layer and the cooling layer are combined into onelayer (heating/cooling layer) creating a heating zone and cooling zone,where the cooling zone is larger than the heating zone. A sample card100 (also termed “RHC card”) may include two thin plates (10, 20) thatsandwich a fluidic sample (90) between them and a heating/cooling layer(112) is under the sample, and the heating/cooling layer (112) is heatedby a heat source positioned away from the card. According to anembodiment, at the edge of the sample, there are no walls to contain thesample, but the edge of the sample will not flow due to capillary forcesthat keep the shape of the fluidic sample edges.

As illustrated in FIG. 25A, plates 10 and 20 may have inner surfaces 11and 21 that are separated by a spacing 102, according to an embodiment.Spacing 102 may be large when the device is ready to receive a sample(e.g., in an open position). FIG. 25B illustrates a closed configurationof device 100 where spacing 102 is made small (e.g., less than about 200μm) to sandwich a sample 90 between plates 10 and 20. In thisembodiment, heating/cooling layer 112 is positioned on an outer surface22 of plate 20.

SH-3 Another detailed description of one embodiment of a RHC card in thepresent invention is that a device for rapidly changing the temperatureof a fluidic sample, comprising:

a first plate (10), a second plate (20), and a heating/cooling layer(112), wherein:

the first plate (10) and the second plate (20) face each other, and areseparated by a distance from each other;

each of the plates has, on its respective inner surface (11, 21), asample contact area for contacting a fluidic sample; wherein the samplecontact areas are facing each other, are in contact with the sample,confines a sample between them, and have an average separation distance(102) from each other, and the sample;

the heating/cooling layer (112) is on the outer surface (22) of thesecond plate (20); and

the heating/cooling layer is configured to comprise a heating zone and acooling zone; wherein the heat zone is configured to heat the fluidicsample, the cooling zone is configured to cool the sample by thermalradiative cooling;

wherein the heating zone is configured to receive heating energy from aheating source and configured to have an area smaller than the totalarea of the heating/cooling layer; and

wherein at least a part of a heating zone of the heating layer overlapswith the sample area.

SH-4 A device for rapidly changing the temperature of a fluidic sample,comprising:

a first plate (10), a second plate (20), a heating layer (112-1), and acooling layer (112-2), wherein:

the first and second plates are movable relative to each other intodifferent configurations;

each of the first plate and the second plate has, on its respectiveinner surface, a sample contact area for contacting a fluidic sample;wherein the sample contact areas face each other, are separated by anaverage separation distance of 200 um or less, and are capable ofsandwiching the sample between them;

the heating layer:

is positioned on the inner surface, the outer surface, or inside of oneof the plates,

is configured to heat a relevant volume of the sample, wherein therelevant volume of the sample is a portion or an entirety of the samplethat is being heated to a desired temperature; and

the cooling layer is:

positioned on the inner surface, the outer surface, or inside of one ofthe plates; and

configured to cool the relevant sample volume; and

comprises a layer of material that that has a thermal conductivity tothermal capacity ratio of 0.6 cm²/sec or larger;

wherein one of the configurations is an open configuration, in which:the two plates are partially or completely separated apart and theaverage spacing between the plates is at least 300 um;

wherein another of the configurations is a closed configuration which isconfigured after the fluidic sample is deposited on one or both of thesample contact areas in the open configuration; and in the closedconfiguration: at least part of the sample is confined by the two platesinto a layer, wherein the average sample thickness is 200 um or less.

SH-5 A device for rapidly changing the temperature of a fluidic sample,comprising:

a first plate (10), a second plate (20), spacers, a heating layer(112-1), and a cooling layer (112-2), wherein:

the first and second plates are movable relative to each other intodifferent configurations;

each of the first plate and the second plate has, on its respectiveinner surface, a sample contact area for contacting a fluidic sample;wherein the sample contact areas face each other, are separated by anaverage separation distance of 200 um or less, and are capable ofsandwiching the sample between them;

one or both of the plates comprise the spacers and the spacers are fixedon the inner surface of a respective plate;

the spacers have a predetermined substantially uniform height that isequal to or less than 200 microns, and the inter-spacer-distance ispredetermined;

the heating layer is:

positioned on the inner surface, the outer surface, or inside of one ofthe plates, and

configured to heat a relevant volume of the sample, wherein the relevantvolume of the sample is a portion or an entirety of the sample that isbeing heated to a desired temperature; and

the cooling layer is:

positioned on the inner surface, the outer surface, or inside of one ofthe plates; and

configured to cool the relevant sample volume; and

comprises a layer of material that that has a thermal conductivity tothermal capacity ratio of 0.6 cm²/sec or larger;

wherein one of the configurations is an open configuration, in which:the two plates are partially or completely separated apart, the spacingbetween the plates is not regulated by the spacers, and the sample isdeposited on one or both of the plates; and

wherein another of the configurations is a closed configuration which isconfigured after the sample is deposited in the open configuration; andin the closed configuration: at least part of the sample is compressedby the two plates into a layer of highly uniform thickness, wherein theuniform thickness of the layer is confined by the sample contactsurfaces of the plates and is regulated by the plates and the spacers.

In some embodiments, the heating/cooling layer (112) can be on the innersurface (21) or inside the second plate (20), rather than on the outersurface (22) of the second plate (20).

In some embodiments of all embodiments of devices, the RHC card furthercomprises spacers that are positioned between the first and second plateto regulate the distance between the two plates (i.e. the spacing of theplates), and hence to regulate the sample thickness. The spacers canallow the thickness of the sample between the two plates uniform over alarge area, even when the plates are thin and flexible.

In some embodiments, there are more than one heating/cooling layer.

A. Small Relevant Sample Volume (RE Ratio)

Reduction of the sample volume that should be heated or cooled to adesirable temperature can shorten the heating time and cooling time in athermal cycle as well as heating power. A reduction of the sample volumethat will be thermal cycled can be achieved by (a) reducing the entiresample volume or (b) heating just a portion of the sample on the sampleholder. The term “relevant sample” or “relevant sample volume” refers tothe volume of the sample that is being heated and/or cooled to desiredtemperatures during a thermal cycling, and the relevant sample can be aportion or an entire volume of a sample on a sample holder, and there isno fluidic separation between the portion of the sample to the rest ofthe sample.

In some embodiments, the relevant volume of the sample is 0.001 ul,0.005 ul, 0.01 ul, 0.02 ul, 0.05 ul, 0.1 ul, 0.2 ul, 0.5 ul, 1 ul, 2 ul,5 ul, 10 ul, 20 ul, 30 uL, 50 ul, 100 ul, 200 ul, 500 ul, 1 ml, 2 ml, 5ml, or in a range between any of the two values.

In some preferred embodiments, the relevant sample volume is in a rangeof 0.001 uL to 0.1 uL, 0.1 um to 2 uL, 2 uL to 10 uL, 10 uL to 30 uL, 30uL to 100 uL, 100 uL to 200 uL, or 200 uL to 1 mL.

In some preferred embodiments, the relevant sample volume is in a rangeof 0.001 uL to 0.1 uL, 0.1 um to 1 uL, 0.1 uL to 5 uL, or 0.1 uL to 10uL.

In certain embodiments, the ratio of the relevant sample to entiresample volume (RE ratio) is 0.01%, 0.05%, 0.1%, 0.5%, 0.1%, 0.5%, 1%,5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or in a rangebetween any of the two values.

In some preferred embodiments, the RE ratio is in a range of between0.01% and 0.1%, 0.1% and 1%, 1% and 10%, 10% and 30%, 30% and 60%, 60%and 90%, or 90% and 100%.

To heat only a portion of the sample, in some embodiments, the area ofthe heating zone is only a fraction of the sample lateral area, and thefraction (i.e. the ratio of the heating zone to the sample lateral area)is 0.01%, 0.05%, 0.1%, 0.5%, 0.1%, 0.5%, 1%, 5%, 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 99%, or in a range between any of the twovalues.

In some preferred embodiments, the ratio of the heating zone area to thesample lateral area is in a range of between 0.01% and 0.1%, 0.1% and1%, 1% and 10%, 10% and 30%, 30% and 60%, 60% and 90%, or 90% and 99%.

B. Local Heating, High Vertical to Lateral Heat Transfer

When a high-K (high thermal conductivity) layer is (e.g. a metal layer)on the inner surface, the outer surface, or inside of one of the platesof a sample holder (RHC card), to make only a part of the high-K layerand a part of sample volume above the part of the high-K layer to beheated to desired temperatures, while keeping the rest of the high-Klayer and the rest of the sample volume at much lower temperaturesduring a thermal cycling, several conditions must be met. The keyconditions are (1) the heat source must directly heat a portion of thehigh-K layer (the portion is termed “heat zone” e.g., only the portionis directly heated by a LED light or has a local electric heater, whilethe rest is not), (2) the vertical heating transfer between the heatzone and a portion of the sample should be much larger than the lateralheat transfer within the high-K material (i.e. in the lateral directionof the high-K material), (3) the relevant sample should have a largelateral to vertical size ratio, and (4) the heating power of the heatzone must sufficient to heat up the relevant sample volume in a timeframe that lateral heat transfer (i.e., heat conduction) is relativelynegligible.

To satisfy the condition (2) above, the scaled thermal conduction ratio(STC ratio) of the vertical heat transfer from the high-K heating zoneto the sample through the middle layer that is between the high-K andthe sample to the lateral heat transfer inside the high-K layer isdefined as:STC ratio=η=0.025·K _(m) K _(s) D ² /K _(k)(K _(m) t _(s) +K _(s) t_(m))t _(k)

wherein K_(k), K_(s), and K_(m) is, respectively, the thermalconductivity of the high-K layer, the relevant sample, and the middlelayer (i.e. the layer between the high-K and the sample), t_(k), t_(s),and t_(m) is, respectively, the thickness of the high-K layer, thesample, and the middle layer; D is the average lateral dimension of therelevant sample, and 0.025 is a scaling factor.

To locally heat a part of the high-K layer and a part of sample volumeabove the part of the high-K layer to desired temperatures, whilekeeping the rest of the high-K layer and the rest of the sample volumeat much lower temperatures during a thermal cycling. In someembodiments, the scaled thermal conduction ratio (STM ratio) is 2 orlarger, 5 or larger, 10 or larger, 20 or larger, 30 or larger, 40 orlarger, 50 or larger, 100 or larger, 1000 or larger, 10000 or larger,10000 or larger, or in a range between any of the two values.

In some preferred embodiments, the scaled thermal conduction ratio (STMratio) is in a range of between 10 to 20, 30 to 50, 100 to 1,000, 1,000to 10,000, or 10,000 to 1,000,000.

To satisfying the condition (2) and (3) above, in some embodiments, thelateral to vertical size (LVS) ratio for relevant sample is 5, 10, 20,50, 70, 100, 200, 300, 400, 500, 600, 700, 800, 800, 1,000, 2,000,5,000, 10,000, 100,000, or in a range between any of the two values.

In some preferred embodiments, the LVS ratio for relevant sample is in arange of 5 to 10, 10 to 50, 50 to 100, 100 to 500, 500 to 1,000, 1,000,to 10,000, or 10,000 to 100,000,

In certain embodiments, the thickness of the relevant sample is reduced(which also can help sample heating speed), and the relevant sample hasa thickness of 0.05 um, 0.1 um, 0.2 um, 0.5 um, 1 um, 2 um, 5 um, 10 um,20 um, 30 um, 40 um, 50 um, 60 um, 70 um, 80 um, 90 um, 100 um, 200 um,300 um, or in a range between any of the two values.

In some preferred embodiments, the relevant sample has a thickness in arange between 0.05 um and 0.5 um, 0.5 um and 1 um, 1 um and 5 um, 5 umand 10 um, 10 um and 30 um, 30 um and 50 um, 50 um and 70 um, 70 um and100 um, 100 um and 200 um, or 200 um and 300 um.

C. Large Sample to Non-Sample Thermal Mass Ratio (NSTM Ratio)

An increase of the sample-to-non-sample thermal mass ratio can shortenheating time, reduce heating energy, and increase energy efficiency. Inan embodiment where a sample is sandwiched between the two plates, athermal mass ratio can be estimated by only considering the relevantsample volume and the portions of the two plates that sandwich therelevant sample, assuming there are no thermal losses in these volumes.Therefore, one parameter to measure a thermal mass ratio is the ratio of“specific area thermal mass” of the relevant sample to the non-sample(the portions of the plates that sandwich the relevant sample as well asthe part heating/cooling layer on the plate portion). The term “specificarea thermal mass” of a material refers to as the volume specific heatof the material multiplying its thickness.

The sample to non-sample thermal mass ratio is a ratio of the usefulheat energy (which directly heat the relevant sample) to the “wastedheat energy (that heats non-sample materials), assuming that the heatlosses by thermal conduction and radiation are negligible.

For examples, water has a volume specific heat of 4.2 J/(cm³·° C.), thusthe area specific heat for a 30 um thick water layer is 1.26×10⁻²J/(cm³·° C.). A PMMA has a volume specific heat of 1.77 J/(cm³·° C.),thus the area specific heat for a 25 um thick PMMA layer is 4.43×10⁻³J/(cm³·° C.), which is ˜2.8 times less than that of 30 um water layer.Gold has a volume specific heat of 2.5 J/(cm{circumflex over ( )}3-C),thus the area specific heat for a 0.5 um thick gold layer is 1.25×10⁻⁴J/(cm{circumflex over ( )}2-C), which is ˜100 times less than that of 30um water layer, and is negligible. The negligible area specific heat ofthe Au is due to its thin thickness.

If, in a RHC card embodiment, the relevant sample is sandwiched betweentwo plates of 25 um thick each and the heating/cooling layer is 0.5 umthick, then the sample to non-sample thermal mass ratio for this case is˜1.4. Namely, when the heat losses by thermal conduction and radiationare neglected, the useful energy to the wasted energy ratio is ˜1.4, andthe useful energy to the total heating energy ratio is 58%.

In some embodiments, the sample to non-sample thermal mass ratio (NSTMratio) is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 1, 1.5, 2, 3, 4, 5, 10, 20,30, 40, 50, 60, 70, 100, 200, 300, 1000, 4000, or in a range between anyof the two values.

In preferred embodiments, the sample to non-sample thermal mass ratio(NSTM ratio) is in a range of between 0.1 to 0.2, 0.2 to 0.5, 0.5 to0.7, 0.7 to 1, 1 to 1.5, 1.5 to 5, 5 to 10, 10 to 30, 30 to 50, 50 to100, 100 to 300, 300 to 1,000, or 1,000 to 4,000.

To make the sample to non-sample thermal mass ratio high, one needs tokeep the area thermal mass of the non-sample low, which in turn, needsto make the plates and the heating/cooling layer thin, and/or the volumespecific heat low.

To make the thermal mass ratio large, one embodiment uses a thinmaterial that has multi-layers or mixed materials. For examples, acarbon fiber layer(s) with plastic sheets or carbon mixed with plastics,which can have a thickness of 0.1 um, 0.2 um, 0.5 um, 1 um, 2 um, 5 um,10 um, 25 um, 50 um, or in a range between any of the two values.

D. Thin Thickness and Large Lateral to Vertical Size Ratio (LVS Ratio)for Relevant Sample

The term of “lateral to vertical size ratio for sample” or “LVS ratiofor sample” refers to the ratio of the average lateral size of therelevant sample volume to its average vertical size. A larger LVS ratiofor sample can reduce the wasted heating energy and increase heatingspeed and/or cooling speed in the embodiments that the heating and/orcooling is primarily from the vertical direction, and can reduce thelateral thermal conduction loss at the edge of the relevant samplerelative to the total thermal energy. All of these can increase and/orcan increase cooling time.

In some embodiments, the LVS ratio for relevant sample is 5, 10, 20, 50,70, 100, 200, 300, 400, 500, 600, 700, 800, 800, 1,000, 2,000, 5000,10,000, 100,000, or in a range between any of the two values.

In some preferred embodiments, the LVS ratio for relevant sample is in arange of 5 to 10, 10 to 50, 50 to 100, 100 to 500, 500 to 1,000, 1000,to 10,000, or 10,000 to 100,000,

For example, a sample has a lateral dimension of 15 mm and a thicknessof 30 um, hence an LVS for the sample of 500.

In certain embodiments, the thickness of the relevant sample is reduced(which also can help sample heating speed), and the relevant sample hasa thickness of 0.05 um, 0.1 um, 0.2 um, 0.5 um, 1 um, 2 um, 5 um, 10 um,20 um, 30 um, 40 um, 50 um, 60 um, 70 um, 80 um, 90 um, 100 um, 200 um,300 um, or in a range between any of the two values.

In some preferred embodiments, the relevant sample has a thickness in arange between 0.05 um and 0.5 um, 0.5 um and 1 um, 1 um and 5 um, 5 umand 10 um, 10 um and 30 um, 30 um and 50 um, 50 um and 70 um, 70 um and100 um, 100 um and 200 um, or 200 um and 300 um.

E. Thin Thickness and Large Lateral to Vertical Size Ratio (LVS Ratio)for Non-Samples

The term of “lateral to vertical size ratio for non-sample” or “LVSratio for non-sample” refers to the ratio of the average lateral size ofthe portions of the two plates that sandwich the relevant sample (whichis the same as the average lateral size of the relevant sample volume)to its thickness. A large LVS ratio for non-sample can reduce thelateral thermal conduction loss at the edge of the non-sample relativeto the total thermal energy.

In some embodiments, the LVS ratio for non-sample is 5, 10, 20, 50, 70,100, 200, 300, 400, 500, 600, 700, 800, 800, 1,000, 2000, 5000, 10,000,100,000, or in a range between any of the two values.

In preferred embodiments, the LVS ratio for non-sample is in a range of5 to 10, 10 to 50, 50 to 100, 100 to 500, 500 to 1,000, 1000, to 10,000,or 10,000 to 100,000,

For example, two 25 um thick plates sandwich a sample of 5 mm or largerlateral dimension of the relevant sample, hence an LVS for thenon-sample of 200 or higher for each plate.

To shorten heating time, reduce heating energy, and increase energyefficiency, the lateral thermal conduction through a non-sample material(on the sample holder) should be reduced.

In particularly, when the first and the second plates are made of thematerials that are not good thermal materials, the thickness of theplates should be minimized.

In some embodiments, the first plate or the second plate or each of bothplates has a thickness of 10 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm,1 um, 2.5 μm, 5 um, 10 um, 25 um, 50 um, 100 um, 200 um, or 500 um, 1000um, or in a range between any of the two values.

In some preferred embodiments, the first plate or the second plate oreach of both plates has a thickness of 10 nm, 100 nm, 200 nm, 300 nm,400 nm, 500 nm, 1 um, 2.5 μm, 5 um, 10 um, 25 um, 50 um, 75 um, or in arange between any of the two values.

The first plate and the second plate can have the same thickness or adifferent thickness, and can be made of the same materials or differentmaterials.

In some preferred embodiments, the first plate or the second plate oreach of both plates has a thickness in a range of between 10 nm and 500nm, 500 nm and 1 um, 1 um and 2.5 um, 2.5 um and 5 um, 5 um and 10 um,10 um and 25 um, 25 um and 50 um, 50 um and 100 um, 100 um and 200 um,or 200 um and 500 um, or 500 um and 1,000 um.

In some preferred embodiments, the first plate and second plates areplastic, a thin glass, or a material with similar physical properties.The first plate or second plate has a thickness of 100 nm, 500 nm, 1 um,5 um, 10 um, 25 um, 50 um, 100 um, 175 um, 250 um, or in a range betweenany of the two values.

In some preferred embodiments, the first plate and second plates areplastic, a thin glass, or a material with similar physical properties.The first plate has a thickness of 5 um, 10 um, 25 um, 50 um, or in arange between any of the two values; while the second plate (that platethat has heating layer or cooling layer) has a thickness of 100 nm, 500nm, 1 um, 5 um, 10 um, in a range between any of the two values.

F. Cooling Layer of High K and/or High Thermal Conductivity-to-CapacityRatio (KC Ratio)

Since any thermal conduction through a non-sample material will wasteenergy and since lateral thermal conduction has much longer thermal paththan vertical thermal conduction, the energy wasted in lateral thermalconduction in non-sample materials should be minimized. One way tominimize this type of wasted energy is to use a high thermal conduction(high-K) or more precisely a high thermal conductivity-to-capacity ratio(KC ratio) materials for the cooling layer. For a given thermalconductivity, a given temperature change, and a given geometry, a high Kand/or a high KC ratio material would need much less energy to be heatedup than a low K and/or low KC ratio material.

In some embodiments, the KC ratio materials for the cooling layer isequal to or higher than 0.1 cm²/sec, 0.2 cm²/sec, 0.3 cm²/sec, 0.4cm²/sec, 0.5 cm²/sec, 0.6 cm²/sec, 0.7 cm²/sec, 0.8 cm²/sec, 0.9cm²/sec, 1 cm²/sec, 1.1 cm²/sec, 1.2 cm²/sec, 1.3 cm²/sec, 1.4 cm²/sec,1.5 cm²/sec, 1.6 cm²/sec, 2 cm²/sec, 3 cm²/sec, or in a range betweenany of the two values.

In some preferred embodiments, the KC ratio for the cooling layer is ina range of between 0.5 cm²/sec and 0.7 cm²/sec, 0.7 cm²/sec and 0.9cm²/sec, 0.9 cm²/sec and 1 cm²/sec, 1 cm²/sec and 1.1 cm²/sec, 1.1cm²/sec and 1.3 cm²/sec, 1.3 cm²/sec and 1.6 cm²/sec.

In some embodiments, a high thermal conductivity (i.e. high-K) materialis used for the cooling layer, and the high-K material has a thermalconductivity that is equal to or larger than 50 W/(m·K), 80 W/(m·K), 100W/(m·K), 150 W/(m·K), 200 W/(m·K), 250 W/(m·K), 300 W/(m·K), 350W/(m·K), 400 W/(m·K), 450 W/(m·K), 500 W/(m·K), 600 W/(m·K), 1000W/(m·K), 5000 W/(m·K), or in a range between any of the two values.

In some preferred embodiments, a high thermal conductivity (i.e. high-K)material is used for the cooling layer, and the high-K material has athermal conductivity that is in the range of 50 W/(m·K) to 100 W/(m·K),110 W/(m·K) to 200 W/(m·K), 200 W/(m·K) to 400 W/(m·K), 400 W/(m·K) to600 W/(m·K), or 400 W/(m·K) to 5000 W/(m·K).

In some embodiments, the high-K material is selected from metals,semiconductors, and allows of thermal conductivity higher than 50W/(m·K), and any combinations (including any mixtures). In someembodiments, the high-K material is selected from gold, copper, silver,and aluminum, and any combinations (including any mixtures). In someembodiments, the high-K material is selected from carbon particles,carbon tubes, graphite, silicon, and any combinations (including anymixtures).

G-1. Cooling Zone Area Larger than Lateral Relevant Sample Area andHeating Zone Area

To effectively cool a sample while reducing the wasted energy innon-sample materials, in some embodiments, a high K and/or a high KCratio material (termed “high K material”) is used as the major channelfor removing the heat from the sample. The area of high-K cooling zone(layer) should be larger than the relevant sample lateral size.

In certain embodiments, the cooling zone (layer) has an area that islarger than the lateral area of the relevant sample by a factor of 1.5,2, 3, 4, 5, 10, 20, 50, 70, 100, 200, 300, 400, 500, 600, 700, 800, 800,1,000, 2,000, 5,000, 10,000, 100,000, or in a range between any of thetwo values.

In preferred embodiments, the cooling zone (layer) has an area that islarger than the lateral area of the relevant sample by a factor in arange of 1.5 to 5, 5 to 10, 10 to 50, 50 to 100, 100 to 500, 500 to1,000, 1000, to 10,000, or 10,000 to 100,000.

To increase the cooling speed and thermal cycling efficiency, in certainembodiments, the high-K cooling layer (zone) should an area to largethan the heating zone area.

In some embodiments, the area of the cooling zone (layer) is larger thanthe area of the heating zone (layer) by a factor (i.e. the ratio of thecooling zone area to the heating zone area, “CH ratio”) of 1.1, 1.5, 2,3, 4, 5, 10, 20, 30, 40, 50, 70, 100, 200, 300, 400, 500, 600, 700, 800,800, 1,000, 5000, 10,000, 100,000, or in a range between any of the twovalues.

In preferred embodiments, the cooling zone (layer) has an area that islarger than the lateral area of the hearing zone (layer) by a factor ina range of 1.1 to 1.5, 1.5 to 5, 5 to 10, to 50, 50 to 100, 100 to 500,500 to 1,000, 1,000, to 10,000, or 10,000 to 100,000.

G-2. Cooling Zone Area and Heating Zone Area are the Same as LateralRelevant Sample Area

In certain embodiments, cooling zone area and heating zone area are thesame as lateral relevant sample area, which is much smaller than thetotal sample area on the plat, and is smaller than the area of theplate. The cooling zone has an area of 1 mm², 1 mm², 1 mm², 1 mm², 1mm², 1 mm², 1 mm², 1 mm², 1 mm², 1 mm², 1 mm², 1 mm², 1 mm², 1 mm²,

The cooling zone can have different shape. In certain embodiments, thereare more than one cooling zones on one plate, and the cooling zones areseparated from each other by a low thermal conductive material such asair or plastic.

H. Heating Zone of High K and/or High Thermal Conductivity-to-CapacityRatio (KC Ratio)

Since any thermal conduction through a non-sample material that willwaste energy and lateral thermal conduction has much longer thermal paththan vertical thermal conduction, the energy wasted in lateral thermalconduction in non-sample materials should be minimized. One way tominimize this type of wasted energy is to use high thermalconductivity-to-capacity (KC) ratio materials for the materials inheating zone, which would need much less energy of heating up for agiven thermal conductivity, a given temperature change, and a givengeometry.

In some embodiments, the KC ratio materials for the heating layer isequal to or higher than 0.1 cm{circumflex over ( )}2/sec, 0.2cm{circumflex over ( )}2/sec, 0.3 cm{circumflex over ( )}2/sec, 0.4cm{circumflex over ( )}2/sec, 0.5 cm{circumflex over ( )}2/sec, 0.6cm{circumflex over ( )}2/sec, 0.7 cm{circumflex over ( )}2/sec, 0.8cm{circumflex over ( )}2/sec, 0.9 cm{circumflex over ( )}2/sec, 1cm{circumflex over ( )}2/sec, 1.1 cm{circumflex over ( )}2/sec, 1.2cm{circumflex over ( )}2/sec, 1.3 cm{circumflex over ( )}2/sec, 1.4cm{circumflex over ( )}2/sec, 1.5 cm{circumflex over ( )}2/sec, 1.6cm{circumflex over ( )}2/sec, 2 cm{circumflex over ( )}2/sec, 3cm{circumflex over ( )}2/sec, or in a range between any of the twovalues.

In some preferred embodiments, the KC ratio for the heating layer is ina range of between 0.5 cm{circumflex over ( )}2/sec and 0.7cm{circumflex over ( )}2/sec, 0.7 cm{circumflex over ( )}2/sec and 0.9cm{circumflex over ( )}2/sec, 0.9 cm{circumflex over ( )}2/sec and 1cm{circumflex over ( )}2/sec, 1 cm{circumflex over ( )}2/sec and 1.1cm{circumflex over ( )}2/sec, 1.1 cm{circumflex over ( )}2/sec and 1.3cm{circumflex over ( )}2/sec, 1.3 cm{circumflex over ( )}2/sec and 1.6cm{circumflex over ( )}2/sec, 1.6 cm{circumflex over ( )}2/sec and 2cm{circumflex over ( )}2/sec, or 2 cm{circumflex over ( )}2/sec and 3cm{circumflex over ( )}2/sec.

In some embodiments, a high thermal conductivity (i.e. high-K) materialis used for the heating layer, and the high-K material has a thermalconductivity that is equal to or larger than 50 W/(m·K), 80 W/(m·K), 100W/(m·K), 150 W/(m·K), 200 W/(m·K), 250 W/(m·K), 300 W/(m·K), 350W/(m·K), 400 W/(m·K), 450 W/(m·K), 500 W/(m·K), 600 W/(m·K), 1000W/(m·K), 5000 W/(m·K), or in a range between any of the two values.

In some preferred embodiments, a high thermal conductivity (i.e. high-K)material is used for the heating layer, and the high-K material has athermal conductivity that is in the range of 50 W/(m·K) to 100 W/(m·K),110 W/(m·K) to 200 W/(m·K), 200 W/(m·K) to 400 W/(m·K), 400 W/(m·K) to600 W/(m·K), or 400 W/(m·K) to 5000 W/(m·K).

In some embodiments, the high-K material is selected from metals,semiconductors, and allows of thermal conductivity higher than 50W/(m·K), and any combinations (including any mixtures). In someembodiments, the high-K material is selected from gold, copper, silver,and aluminum, and any combinations (including any mixtures). In someembodiments, the high-K material is selected from carbon particles,carbon tubes, graphite, silicon, and any combinations (including anymixtures).

To receive light energy by a heating zone (layer), a thermal radiationenhancement surface(s) will be used (on one side or both side of theheating zone). A thermal radiation absorption enhancement surface can beachieved by directly modify the structures of the surface (e.g.patterning nanostructures), coating a high thermal radiation materials(e.g. coating a black paint), or both.

The thermal radiation enhancement surface has a high average lightabsorptance (e.g. the black paint used in our experiments). In certainembodiments, the heating zone has a surface that has an average lightabsorptance of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, or in arange between any of the two values.

In certain preferred embodiments, the heating zone has a surface thathas an average light absorptance in a range of 30% to 40%, 40% to 60%,60% to 80% to 90%, or 90% to 100%.

In some preferred embodiments, the heating zone has a surface that hasan average light absorptance in a range of 30% to 100%, 50% to 100%, 70%to 100%, or 80% to 100%.

In certain embodiments, the heating zone has a surface that has anaverage light absorptance of a value given above by averaging over awavelength range 400 nm to 800 nm, 700 nm to 1500 nm, 900 nm to 2000 nm,or 2000 nm to 20000 nm.

Increasing Thermal Radiative Cooling

In certain embodiments, a fast temperature cycling is achieved byincreasing thermal radiative cooling percentage in the total cooling ofthe sample and the sample holder (i.e. removing heat to the environment)during a thermal cycling, preferably through using high thermalconductivity material as the material for thermal radiative cooling. Onereason is that cooling through lateral thermal conduction needs to heatup many non-sample materials, wasting energy. Another reason is thatthermal radiation cooling is proportional to the fourth power of thetemperature and can be more effective than thermal conduction in a thinfilm.

To enhancing thermal radiative cooling, in certain embodiments, thethermal radiative cooling uses a cooling layer (cooling zone) that isenhanced for thermal radiative cooling. The enhancement includes (i)increase thermal conductivity of the cooling zone (layer), (ii)enlarging the area of the cooling zone (layer), (iii) enhance thesurface thermal radiation of the cooling zone, and (iv) a combinationthereof.

Examples of a high thermal conductivity materials are metals (such asgold, silver, coper, aluminum), semimetals, semiconductors (e.g.silicon) or a combination thereof.

To further enhance thermal radiation of a cooling zone (layer), athermal radiation enhancement surface(s) will be used (on one side orboth side of the cooling zone). A thermal radiation enhancement surfacecan be achieved by directly modify the structures of the surface (e.g.patterning nanostructures), coating a high thermal radiation material(e.g. coating a black paint), or both.

The thermal radiation enhancement surface has a high average lightabsorptance (e.g. the black paint used in our experiments). In certainembodiments, the cooling zone has a surface that has an average lightabsorptance of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, or in arange between any of the two values.

In certain preferred embodiments, the cooling zone has a surface thathas an average light absorptance in a range of 30% to 40%, 40% to 60%,60% to 80% to 90%, or 90% to 100%.

In some preferred embodiments, the cooling zone has a surface that hasan average light absorptance in a range of 30% to 100%, 50% to 100%, 70%to 100%, or 80% to 100%.

In certain embodiments, the cooling zone has a surface that has anaverage light absorptance of a value given above by averaging over awavelength range 400 nm to 800 nm, 700 nm to 1,500 nm, 900 nm to 2,000nm, or 2,000 nm to 20,000 nm.

In certain embodiments, the surface thermal radiation enhancement layeris black paint, plasmonic structures, nanostructures, or any combinationthereof.

The high thermal radiation materials are polymer mixtures that lookblack by human eyes (often termed “black paints”). A high thermalradiation material include, but not limited to, a mixture of polymersand nanoparticles. One example of the nanoparticles is black carbonnanoparticle, carbon, nanotubes, graphite particles, graphene, metalnanoparticles, semiconductor nanoparticles, or a combination thereof.

The high thermal radiation material further comprises a material that isdeposited or made on the layer surface and look blacks by human eyes.The materials include, but not limited to, black carbon nanoparticle,carbon, nanotubes, graphite particles, graphene, metal nanoparticles,semiconductor nanoparticles, or a combination thereof.

The plasmonic structures include nanostructured plasmonic structures.

In some embodiments, a cooling layer comprise a layer of high thermalconductivity metal (50 W/(m·K) or higher) with a surface thermalradiation enhancement layer. In some embodiments, the surface thermalradiation enhancement layer has a low lateral thermal conductance, whichis due to either ultrathin layer, low thermal conductivity, or both.

Percentage of Thermal Radiative Cooling.

In certain embodiments, thermal radiative cooling is achieved byincreasing the area of radiative cooling layer (i.e. a high-K material,unless stated otherwise), and the radiative cooling layer area is largerthan the lateral area of the relevant sample by a factor of 1.2, 1.5, 2,3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80 100, 200, 300, 400, 500, 600,700, 800, 800, 1,000, 2,000, 5,000, 10,000, 100,000, or in a rangebetween any of the two values.

In preferred embodiments, the radiative cooling zone (layer) has an areathat is larger than the lateral area of the relevant sample by a factorin a range of 1.2 to 3, 3 to 5, 5 to 10, 10 to 50, 50 to 100, 100 to500, 500 to 1,000, 1,000, to 10,000, or 10,000 to 100,000.

In some embodiments, the ratio of the thermal radiation cooling by thecooling zone (layer) to the total cooling of the sample and sampleholder during a thermal cycling is 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 99%, or in a range between any of the two values.

In some preferred embodiments, the ratio of the thermal radiationcooling by the cooling zone (layer) to the total cooling of the sampleand sample holder during a thermal cycling is in a range of between 10%and 20%, 20% and 30%, 30% and 40%, 40% and 50%, 50% and 60%, 60% and70%, 70% and 80%, 80% and 90%, or 90% and 99%.

J. Control of Cooling Layer Thickness

In certain embodiments, the thickness of the cooling layer thickness isconfigured to facilitate to optimize heating locally and/or energyefficiency. If the cooling zone (layer) is too thick, a significantpercentage of the heating energy will be wasted by the cooling layer,lengthening heating time (for a given heating power). On the other hand,if the cooling zone is too thin, the cooling time will be significantlylonger. Hence, the cooling layer thickness should be optimized for bothfast heating and cooling.

Through our experiments, we found that the thickness of the high-Kcooling layer can regulate the cooling rate. By selecting a properhigh-K cooling layer thickness and a proper LED power density, a fastheating and cooling can be achieved.

Since a thermal conductance of a layer proportional to a material'sthermal conductivity times the layer thickness, so it is this productshould be optimized.

In some embodiments, a cooling zone (layer) has thermal conductivitytimes its thickness of 6×10⁻⁵ W/K, 9×10⁻⁵ W/K, 1.2×10⁻⁴ W/K, 1.5×10⁻⁴W/K, 1.8×10⁻⁴ W/K, 2.1×10⁻⁴ W/K, 2.7×10⁻⁴ W/K, 3×10⁻⁴ W/K, 1.5×10⁻⁴ W/K,or in a range between any of the two values.

In some preferred embodiments, a cooling zone (layer) has thermalconductivity times its thickness in a range of 6×10⁻⁵ W/K to 9×10⁻⁵ W/K,9×10⁻⁵ W/K to 1.5×10⁻⁴ W/K, 1.5×10⁻⁴ W/K to 2.1×10⁻⁴ W/K, 2.1×10⁻⁴ W/Kto 2.7×10⁻⁴ W/K, 2.7×10⁻⁴ W/K to 3×10⁻⁴ W/K, or 3×10⁻⁴ W/K to 1.5×10⁻⁴W/K.

In certain preferred embodiments, a cooling zone (layer) has thermalconductivity times its thickness in a range of 9×10⁻⁵ W/K to 2.7×10⁻⁴W/K, 9×10⁻⁵ W/K to 2.4×10⁻⁴ W/K, 9×10⁻⁵ W/K to 2.1×10⁻⁴ W/K, or 9×10⁻⁵W/K to 1.8×10⁻⁴ W/K.

In one embodiment, a cooling zone comprises a gold layer of a thicknessin the range of 200 nm to 800 nm. In another embodiment, a cooling zonecomprises a gold layer of a thickness in the range of 300 nm to 700 nm.

K. Large Conductance Between Sample and Heating Zone or Cooling Zone

For a fast heating and cooling a sample, the thermal conduction per unitarea between a relevant sample and a heating layer and/or the coolinglayer should be large. The thermal conduction per area is equal to theconductivity (unit volume) divided by the material thickness for thematerials that are between the HC layer and the sample. For example, for100 nm thick of PS as the second plate which has the HC layer on onesurface and the sample on the other surface, the conductance between theHC layer and the sample is ˜1000 W/(m²·K)

Based on experiments, in some embodiments of a RHC card, the materialsbetween the heating zone and the relevant sample has a thermalconductivity and a thickness configured to be about 1000 W/(m²·K) orhigher.

In some embodiments of a RHC card, the materials between the heatingzone and the relevant sample has a thermal conductivity and a thicknessconfigured to have a conductance per unit area that is equal to orlarger than 1000 W/(m²·K), 2000 W/(m²·Km²·K), 3000 W/(m²·Km²·K), 4000W/(m²·Km²·K), 5000 W/(m²·Km²·K), 7000 W/(m²·Km²·K), 10000 W/(m²·K),20000 W/(m²·K), 50000 W/(m²·K), 50000 W/(m²·K), 100000 W/(m²·K), or in arange of any the values.

A preferred conductance per unit area of the material between theheating zone and the relevant sample is in a range of 1000 W/(m²·K) to2000 W/(m²·K), 2000 W/(m²·K) to 4000 W/(m²·K), 4000 W/(m²·K) to 10,000W/(m²·K), or 10000 W/(m²·K) to 100000 W/(m²·K).

In another preferred embodiment, it has zero distance between theheating zone and the relevant sample, and hence an infinity for theconductance per unit area of the material between the heating zone andthe relevant sample.

In certain embodiments, the heating layer or the cooling layer isseparated from a relevant sample by a thin plastics plate (or film)which has a thermal conductivity in the range of 0.1 to 0.3 W/(m·K), andthe thin plastic layer has a thickness of 0 nm, 10 nm, 50 nm, 100 nm,200 nm, 300 nm, 400 nm, 500 nm, 1 um, 2.5 um, 5 um, 10 um, 25 um, 50 um,75 nm 100 um, 150 um, or in a range between any of the two values

In some preferred embodiments, the thin plastic plate (or film) thatseparate the relevant sample from the heating layer or the cooling layerhas thickness in a range between 0 nm and 100 nm, 100 nm and 500 nm, 500nm and 1 um, 1 um and 5 um, 5 um and 10 um, 10 um and 25 um, 25 um and50 um, 50 um and 75 um, 75 um and 100 um, or 100 um and 150 um.

In one preferred embodiment of the RHC card, the thin plastic plate (orfilm) that separate the relevant sample from the heating layer or thecooling layer has thickness of 1 nm, 10 nm, 0.1 um, 0.5 um, 1 um, 5 um,10 um, 20 um, 25 um, or a range between any two values.

L. Small Relative Reagent Lateral Diffusion

In order to make a biochemical reaction substantially uniform in therelevant sample volume during a temperature change or a thermal cycling,the average lateral area of the relevant sample should be significantlylarger than the lateral diffusion of the nucleic acids and/or otherregents used for a molecular amplification and/or reaction. In this way,during the time of temperature change or a thermal cycling, most of themolecules inside the relevant sample volume do not have enough time todiffuse out of the relevant sample volume, while most of the moleculesoutside the relevant sample volume do not have enough time to diffuseinto the relevant sample volume.

Considering a thermal cycling time duration of 3 min and a diffusionconstant of ˜1×10{circumflex over ( )}-6 cm2/s for a molecule about 600Da molecular weight, the diffusion length is ˜130 um.

In certain embodiments, the ratio of the average lateral size of therelevant sample volume to the diffusion length of the reagent during thetime for thermal cycling or a reaction is equal to or larger than 5, 6,7, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, 1000,5000, 10000, 100000, or in a range between any two values.

In some preferred embodiments, the ratio of the average lateral size ofthe relevant sample volume to the diffusion length of the reagent duringthe time for thermal cycling or a reaction is in a range of 5 to 10, 10to 30, 30 to 60, 6 to 100, 100 to 200, 200 to 500, 500 to 1000, 1000 to5000, 5000 to 10,000, or 10,000 to 100,000.

In some preferred embodiments, the ratio of the average lateral size ofthe relevant sample volume to the diffusion length of the reagent duringthe time for thermal cycling or a reaction is in a range of 5 to 10, 10to 30, 30 to 60, 6 to 100, 100 to 200, 200 to 500, 500 to 1,000, 1,000to 5,000, 5,000 to 10,000, or 10,000 to 100,000.

In certain preferred embodiments, the average lateral dimension of therelevant volume is 1 mm, 2 mm, 3 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm 10 mm,12 mm, 15 mm, 20 mm, 30 mm, 40 mm, 50 mm, 70 mm, 100 mm, 200 mm, or in arange between any two values.

In some preferred embodiments, the average lateral dimension of therelevant volume is in a range of 1 mm to 5 mm, 5 mm to 10 mm, 10 mm to20 mm, 20 mm to 40 mm, 40 mm to 70 mm, 70 mm to 100 mm, or 100 mm to 200mm.

In another preferred embodiments, the average lateral dimension of therelevant volume is in a range of 1 mm to 5 mm, 1 mm to 10 mm, or 5 mm to20 mm.

M. Without Edge Sealing or Simple Edge Sealing

To simplify the sample holder operation and cost, in certainembodiments, there is no sealing between the two plates that confine asample; namely, the sample sandwiched between the plates can evaporatefrom the sample edge into environment. However, in our experiments, wefound that in our sample card configuration, such evaporation isnegligible relative to total sample volume, due to a large ratio of thelateral sample area to the sample edge area; the plates have preventedmost of the evaporation.

In some embodiments, an enclosure ring spacer or some discontinuousspacer walls can be put on one or both of the plates to reduce oreliminate a sample evaporation.

P-2 Forced Air Cool

In certain embodiments, there is a forced air cooling/circulating systemnear the RHC card to speed up the cooling process. The example of forcedair cooling system includes but not limit to a fan circulating the coolair near the card, several fans circulating the cool air near the card,a cooling source cool the air near the card, a cooling pad direct touchthe card or their combinations.

In certain embodiments, there is a forced air cooling/circulating systemcooling the air on the top surface of the card.

In certain embodiments, there is a forced air cooling/circulating systemcooling the air on the bottom surface of the card.

In certain embodiments, there is a forced air cooling/circulating systemcooling the air surrounding all the surface of the card.

2. Mechanical Structure Designs

N. Movable Plates and Compressed Open Flow, Hinges, Opening Notches,Recessed Edge and Sliders

To load a sample simply, in certain embodiments in the presentinvention, the two plates of a RHC card are movable relative to eachother into different configurations. A sample is deposited at an openconfiguration of the plates, and then the plates are pressed into aclosed configuration. During the pressing, the sample will flow betweenthe plates into a thin layer, and the flow is termed “compressed openflow”, since there are plenty room between the plates that allow thesample to flow.

In certain embodiments, spaces for regulating the sample thickness areadded on one or both of the plates, hence a device for rapidly changingthe temperature of a fluidic sample, comprising:

a first plate (10), a second plate (20), a heating layer (112-1), and acooling layer (112-2), wherein:

the first and second plates are movable relative to each other intodifferent configurations;

each of the first plate and the second plate has, on its respectiveinner surface, a sample contact area for contacting a fluidic sample;wherein the sample contact areas face each other, are separated by anaverage separation distance of 200 um or less, and are capable ofsandwiching the sample between them;

the heating layer is:

positioned on the inner surface, the outer surface, or inside of one ofthe plates, and

configured to heat a relevant volume of the sample, wherein the relevantvolume of the sample is a portion or an entirety of the sample that isbeing heated to a desired temperature; and

the cooling layer is:

positioned on the inner surface, the outer surface, or inside of one ofthe plates;

configured to cool the relevant sample volume; and

comprises a layer of material that that has a thermal conductivity tothermal capacity ratio of 0.6 cm²/sec or larger;

wherein one of the configurations is an open configuration, in which:the two plates are partially or completely separated apart and theaverage spacing between the plates is at least 300 um;

wherein another of the configurations is a closed configuration which isconfigured after the fluidic sample is deposited on one or both of thesample contact areas in the open configuration; and in the closedconfiguration: at least part of the sample is confined by the two platesinto a layer, wherein the average sample thickness is 200 um or less;and

wherein, in some embodiments, the heating layer and cooling layer arethe same material layer that has a heating zone and a cooling zone, andwherein the heating zone and cooling zone can have the same area ordifferent areas.

In some embodiments, the sample holder (also termed “RHC card” or“Q-card”) with movable plates further comprises hinges, notches,recesses, which help to facilitate the manipulation of the sample holderand the measurement of the samples. Furthermore, the sample holders canslide into sliders. The structure, material, function, variation anddimension of the hinges, notches, recesses, sliders and compress openflow are herein disclosed, or listed, described, and summarized in PCTApplication (designating U.S.) Nos. PCT/US2016/045437 andPCT/US0216/051775, which were respectively filed on Aug. 10, 2016 andSep. 14, 2016, U.S. Provisional Application No. 62/456,065, which wasfiled on Feb. 7, 2017, U.S. Provisional Application No. 62/456,287,which was filed on Feb. 8, 2017, and U.S. Provisional Application No.62/456,504, which was filed on Feb. 8, 2017, all of which applicationsare incorporated herein in their entireties for all purposes.

Spacers (13)

In certain embodiments, the spacers as described in embodiment SH-5 willbe used to regulate the sample thickness and make the thickness uniform.The spacers also allow to achieve uniform sample thickness, even whenboth plates are very thin (e.g. 25 um thick or less).

In certain embodiments, the spacers are fixed on one or both of theplates. In certain embodiments, the spacers are mixed with the sample.In some embodiments, the spacers have a uniform height and the spacers,together with the first plate and the second plate, regulate the samplelayer. In some embodiments, the thickness of the sample layer issubstantially equal to the height of the spacers.

In some embodiments, the plates are flat (e.g. as shown in FIG. 33A). Insome embodiments, either one or both of the plates include wells (e.g.as shown in FIG. 33B). For example, in certain embodiments the width ofthe wells can be less than 500 um, 200 um, 100 um, 50 um, 25 um, 10 um,5 um, 2.5 um, 1 um, 500 nm, 400 nm, 300 nm, 200 nm, or 100 nm, or in arange between any of the two values. In certain embodiments, the depthof the wells can be less than 500 um, 200 um, 100 um, 50 um, 25 um, 10um, 5 um, 2.5 um, 1 um, 500 nm, 400 nm, 300 nm, 200 nm, 100 nm, 50 nm,20 nm, 10 nm, 5 nm, 2 nm, or 1 nm, or in a range between any of the twovalues

In some embodiments, one or both of the plates have wells and most orentire of the samples are only inside the well of one plate and iscovered by other plate (not shown in the figures).

P. Sample Cartridge and Thermal Conduction Isolation

In certain embodiments, the RHC card (sample holder) can be furthermounted on a sample cartridge. The cartridge can be configured to slidein or out a base (also termed “adaptor”). A base houses the powersource, temperature sensors and controllers, signal measurement devices,and a slot for the sample holder with or without a cartridge to slide inor out of the base.

In some embodiments, the sample holder, the cartridge (i.e. the sampleholder support) or both are “thermal conduction isolated”, namely, theydo not have or almost do not have, during a thermal cycling, a thermalconduction to the environment. In this case, the cooling in the thermalcycling is essentially by thermal radiation (this is termed “noconductive heat transfer”). In some embodiment, the “thermal conductionisolation” is achieved in the sample holder, the cartridge, or both byconfiguration their materials, the geometry (including of thicknessreduction), or both.

Q. Combination of Above

An embodiment of a RHC card can be any combination of the specificationdescribed in SH-1, SH-2, SH-3 and in subsections of A to P.

R. Heating Sources

The heating layer or the heating/cooling layer in a RHC card isconfigured to be heated by a heating source, wherein the heating sourcedelivers heat energy to the heating/cooling layer optically,electrically, by radio frequency (RF) radiation, or a combinationthereof.

S. Base (i.e. Adaptor)

In some embodiments, the apparatus further comprises a base (an adaptor)that is configured to house the sample card, the heating source,temperature sensors, a part of an entire of temperature controlled(include a smartphone in some embodiments), extra-heat sink(optionally), a fan (optionally) or a combination of thereof. In someembodiments, the adaptor comprises a card slot, into which the samplecard or a sample cartridge can be inserted. In some embodiments, thesample card or the sample cartridge, after being fully inserted into theslot, or after reaching a pre-defined position in the slot, isstabilized and stays in place without any movement.

T. Smartphone

In some embodiments, a smartphone is used to mage the sample card,controlling the heating and/cooling, sensing a signal, monitor operationuse camera, provide light/energy with a flash, communicate to a local ora remote device, integrated through a base (adaptor) in a system, of acombination thereof.

U. Applications for Isothermal Nucleic Acid Amplification

The present invention with a slight modification also provides usefuldevices and methods for isothermal nucleic acid amplification, where asample temperature needs to be raised from environment to an elevatedtemperature (i.e. 65° C.) and keep at the temperature for a period oftime (i.e. 5-10 min). In some embodiments, one of the modificationsneeded for isothermal nucleic acid amplification test, is to reduce oreliminate the cooling zone/layer, so that loss of thermal energy fromthe sample and/or the sample holder to the environment is reduced.

The present invention with a slight modification provides useful devicesand methods for reverse transcription polymerase chain reaction, whichcontains an isothermal process before the regular PCR, where a sampletemperature needs to be raised from environment to an elevatedtemperature (i.e. 50° C.) and keep at the temperature for a period oftime (i.e. 5-10 min). The present invention with a slight modificationprovides useful devices and methods for minimize PCR cross-contaminationas method to use dUTP and uracil-DNA N-glycosylase, where a sampletemperature needs to be raised from environment to an elevatedtemperature (i.e. 50° C.) and keep at the temperature for a period oftime (i.e., 1-20 min).

Experimentations of Certain Embodiments

Certain embodiments of the present invention have been testedexperimentally. Some of experimental results are illustrated here.

In some of our experiments, the apparatus, illustrated in FIG. 39A,comprises a sample holder (e.g. an RHC card), a LED light source (i.e.energy source) that was focused by a lens onto an area of (˜5 mm by 5mm) of the sample holder, and a sample holder support (not shown in FIG.39A) made of a thermal conduction insulating material. The sample holdersupport supports a ˜2 mm rim at the two opposite edges of the secondplate (e.g., around a perimeter of the second plate.) There was no extraheat sink, and the heat was mainly radiated into an open environment(e.g., the room).

In the experiments described in this section, the sample holdercomprises a first plate, a second plate, and a heating/cooling layer.One of the plates has spacers. The first plate and second plates aremovable relative to each other into different configurations. One of theconfigurations is an open configuration, wherein the two plate areseparated an average distance at least 300 um. In an open configuration,a sample deposited on one of the plates. Then the other card was placedon top of the sample, and a hand pressing of the two plates into a closeconfiguration. In the close configuration, the spacers regulate thedistance between the two plates, and therefore the sample thickness isregulated by the two plates and the spacers. By using proper spacers andplates (see other part of the description), the sample thickness at aclosed configuration can be uniform over large area and is close to thespacer height. Experimentally, we found that the sample thickness wasuniform even different hand pressing forces, pressures and sequence(pressing one area first and then rub into other area of the RHC card).

The first plate is made of a poly(methyl methacrylate) (PMMA) film of˜50 um thickness, 20 mm wide and 20 mm long.

The second plate is a polyethylene terephthalate (PET) film of 20 mmwide square and 25 um thickness. The second plate has, on its innersurface, a periodic array of pillar spacers of 30 um height, 30 um×40 umsize and 80 um inter spacer distance. The spacer has a uniform heightand a flat top surface (Note other types of spacers can be used and willbe described later). The spacers, that are fixed on the plate, werefabricated by a direct imprint of the flat PMMA plate (other fabricationmethods are also possible).

Various heating/cooling layer of different materials and geometries oneither outer or inner surface of the second plate were experimentallytested. One example (shown in FIG. 39A) is that the heating/coolinglayer is on the outer surface of the second plate, and covers the entiresecond plate outer surface. The heating/cooling layer comprises an Au(gold) film and a black paint layer. The gold film has one surface incontact with the second plate outer surface, and another surface beingpainted with a black paint. The black paint is a commercial product of afilm composited of black carbon nanoparticle and polymer mixture. Theblack paint had an average thickness of ˜9 um (˜2 um thicknessvariation). The black paint layer may be directly facing incoming LEDlight as illustrated in FIG. 40 . Between the Au film and the secondplate outer surface, there is a 5 nm adhesion layer of Ti, whichimproves the adhesion between Au and the second plate. But the adhesionlayer is optional and should have minor or no effects on thermalproperties of the sample holder, because of the thin thickness of theadhesion layer.

The heating source may be a blue light emitting diode (LED) with acentral wavelength of 450 nm. As illustrated in FIG. 40 , light from theLED was projected, using a lens, to the heating/cooling layer, but onlyon the central area of the heating/cooling layer, and typically the LEDspot of size (i.e. area) on the heating/cooling layer is about 5 mm×5mm, according to some embodiments. As shown later, for the given samplecard and the sample thickness, only the sample over the LED heating spotcan change and/or reach the designed temperature. Hence the heating zonearea is about 5 mm×5 mm. In some embodiments, compared with the coolingzone area which is the entire area of the first plate, the cooling zonearea may be about 16 times larger than that of the heating zone area(i.e. high-K C/H ratio=16).

The LED heating source is powered by a power supply that can change theLED current with a time less than 100 ms. In some embodiments, anaspherical condenser lens is used to focus the LED light and the lenshas a diameter of 12 mm, focal length of 10.5 mm and numerical aperture(N.A.) of 0.54.

A temperature sensitive dye (LDS698) monitors the temperature of thesample in the heating zone (i.e. the area directly radiated by LED). Thephotodetector was used to monitor the temperature sensitive dye andfeedback to control the LED current and hence the LED heating source andthe heating zone temperature.

In the experiments described below (Experiment 1 to Experiment 12),unless stated otherwise, the sample holder supports the sample holder bysupporting a ˜2 mm rim at the two opposite edges of the second plate,therefore the sample holder is thermal conduction isolated from theoutside, the cooling of sample holder is primarily by thermal radiativecooling. The thermal radiative cooling is primarily provided by the H/Clayer, since the sample and the plates are poor thermal radiator andhave much lower thermal conduction than the H/C layer. The thermalradiative cooling radiates thermal energy into an open environment (i.e.the room).

In our experiments, ˜5 uL liquid sample with thermal properties close towater was deposited between the two plates and approximately in thecenter area of the plate surface. The sample was dropped on one of theplates of a RHC card first, then the other card was placed on top of thesample, and a hand press of the two plates into a closed configuration.Due to the spacers on the plate, when the two plates are in a closedconfiguration, the spacing between the two plates are regulated by the30 um height spacer array to a separation of 30 um, and the samplethickness was found uniform even with different hand pressing forces,pressures and sequence (pressing one area first and then rub into otherarea of the RHC card). To achieve a good sample thickness by human handpressing offers several advantages in practical use of the presentinvention.

For a ˜5 uL sample between the two plates, the sample has a thickness of30 um and an area of ˜166 mm{circumflex over ( )}2 (approximately ˜13 mmby ˜13 mm square—the sample lateral shape is influenced by the spacerson the plate as illustrated in the top-down view of FIG. 39B). In someembodiments, the total sample area is over ˜6.6 times larger than theheating zone area (˜5 mm by 5 mm). Experimentally, we found that withthis setup, only the potation of the sample above the heating zone gotheated to the desired temperature. Namely, the area (volume) of theportion of the sample being heated is about ⅙th of the total sample area(volume).

In this setup, there was no physical wall at the edge of the sampledisk, but only air. However, as described later, we found that thesample disk diameter before and after a 30 cycle PCR does not changemuch (i.e. hardly seen the difference by a naked eye), this means thateven without a physical wall (except an air-liquid interface) to encloseliquid sample, the sample evaporation is nearly negligible.

All spacers used in this experimental section are the pillars fixed onone plate and having a flat top that can contact the other plate.

In our experiments, the liquid sample was deposited on one of the plateand then the second plate was put on top of the sample. The plates werepressed together by human hands. During the hand pressing, the samplespreads to form a film between the plates. Due to the spacers (ofuniform height) on the plate, even with a hand-pressing, the finalsample thickness is uniform and regulated by the two plate surfaces andthe spacer height. Furthermore, after the sample reaches the finalthickness and the hand pressing force was removed, the two plates of thesample holder “self”-hold to each other by the capillary force of theliquid sample to “self”-maintain the constant sample thickness.Moreover, even during a thermal cycling of 65-95 C, the capillary forcestill held the sample thickness constant. Such self-sample holdingwithout using any clamps can greatly simplify the device operation andcost.

Experiment 1 Light Absorption of Different H/C Layer Materials

In this experiment, the effects of the materials for H/C layer onabsorption of the LED light were studied.

Optical absorption spectrum of different materials for heating/cooling(H/C) layer. Experimentally, we tested the optical absorption spectrum(i.e. 1-R (the light reflection)) of four different H/C layer materialsfor the 450 nm LED irradiation: Au (gold) only (i.e. without a blackpaint) of 500 nm thick, Al (aluminum) only of 400 nm thick, Au (500 nmthick) with a black paint (9 um thick), and Al (400 nm thick) with ablack paint (9 um thick). We found that as shown FIG. 41 , the opticalabsorption is ˜99% for the black paint coated Au and A1 for the entirewavelength range of 400 to 800 nm, a maximum 73% (at ˜490 nm wavelength)and much smaller after 490 nm wavelength for the Au only; and 0.1% over400 nm to 800 nm bandwidth for Al only. This means that the 9 um thickblack paint used in our experiments has greatly enhanced the lightabsorption and radiation of the H/C layer.

Experiment 2 Heating Zone Area Size Measurements

In this experiment, the area of heating zone on the HC layer wasmeasured. We found experimentally that due to the fact that verticalheat transfer from the HC layer to the plates and sample are severalorders of the magnitude better than the lateral thermal conduction inthe plates, sample even with HC layer. The area of heating zone in thesample is about the same area as the LED irradiation area on the HClayer.

Experimentally, the sample holder card (as shown in FIG. 39A) has afirst plate of 50 um thick PMMA plate, a second plate of 25 um thickPET, 30 um thick sample gap controlled by spacers, and a H/C layer ofgold is on the outer surface of the second plate. The first plate, thesecond plate, and the gold/black-paint HC layer have the same area of 20mm×20 mm. The HC layer comprises an Au (gold) film of 500 nm thick and ablack paint layer. The gold film has one surface in contact with thesecond plate outer surface, and another surface being painted with ablack paint. The black paint is a commercial product of a filmcomposited of black carbon nanoparticle and polymer mixture. The blackpaint had an average thickness of ˜9 um (˜2 um thickness variation). TheLED heating power projected on ˜5 mm×5 mm heating zone of the H/C layeris 300 mW. The sample liquid is 5 uL temperature sensitive dye LDS698 2mg/mL in 60% water and 40% DMSO. The temperature sensitive dye allows usto measure the sample temperature optically. The 5 uL sample on the RHCcard has 30 um thickness and ˜167 mm² area, which is much larger thanthe heating zone area. The thermal cycling is between 65° C. and 95° C.

We have observed experimentally that for the given condition, in thermalcycling (65-95° C.), based on measuring temperature sensitive dye, forthe 167 mm² of sample area, only the sample area on top of the LEDdirect irradiation (˜5 mm×5 mm) has a thermal cycling (65-95° C.), whilethe rest of the sample area stays nearly a constant temperature close tothe room temperature (i.e. the environment temperature (e.g. ˜20° C.)).The thermal cycling zone in the sample is approximately about ⅙th of thetotal sample area. The transition distance from the thermal cycling zoneof the sample to the sample area with the environment temperature is,measured from the temperature sensitive dye, approximately 2-3 mm. Thisexperiment also indicates that for the gold/black-paint HC layer of 20mm×20 mm area, only the area of sample that is directly irradiated bythe LED (˜5 mm×5 mm) is heated. Namely, the heating zone is only1/16^(th) of the total HC layer (i.e. high-K C/H ratio=16). The reasonis, as stated before, that in the given RHC card, the vertical heattransfer from the HC layer to the plates and sample are several ordersof the magnitude better than a lateral thermal conduction in the platesand sample with HC layer.

Experiment 3 HC Layer Area Effects on Heating and Cooling Time

In this experiment, the effects of H/C zone area on heating and coolingtime were studied. Two types of RHC cards were investigated.

Type-1 RHC card uses round disk-shaped HC layer. Type-1 RHC cards mayinclude a first plate of 100 um thick PMMA poly(methyl methacrylate)plate, a second plate of 50 um thick PET (polyethylene terephthalate),30 um thick sample gap controlled by spacers, and a H/C layer is 700 nmthick gold film on the outer surface of the second plate. The firstplate and the second plate have a square shape and the same area of 20mm×20 mm. The second plate has, on its inner surface, a periodic arrayof pillar spacers of flat top, uniform 30 um height, 30 um×40 um sizeand 80 um inter spacer distance. The HC layer positioned at center ofthe outer surface of the second plate is an Au layer of 700 nm thicknessand a round disk shape with different disk diameters for different RHCcards.

Type-2 RHC card uses a square shaped HC layer. Type-2 RHC cards mayinclude a first plate of 50 um thick PMMA plate, a second plate of 50 umthick PET, 30 um height spacers to control a sample thickness to 30 um,and a H/C layer is a 500 nm thick gold film on the outer surface of thesecond plate. The first plate has a square shape, an area of 20 mm×20mm, and, on its inner surface, a periodic array of pillar spacers offlat top, uniform 30 um height, 30 um×40 um size and 80 um inter spacerdistance. The second plate has a square shape, and four different areafor four different HC layers. Two of the second plates have area of 20mm×20 mm for the HC layer area of 10 mm×10 mm and 20 mm×20 mm,respectively; but the other two have area same as the HC layer for theHC layer area of 30 mm×30 mm and 40 mm×40 mm, respectively.

In testing two types of RHC cards, the LED heating power projected on ˜5mm×5 mm area of the H/C layer to form a heating zone and has a power of300 mW. The sample liquid is 5 uL temperature sensitive dye LDS698 2mg/mL in 60% water and 40% DMSO. The temperature sensitive dye allows usto measure the sample local temperature optically. The 5 uL sample onthe RHC card has 30 um thickness (regulated by the spacer) and ˜167mm{circumflex over ( )}2 area, which is much larger than the heatingzone area. The thermal cycling is between 65° C. and 95° C.

The experimental data (shown in FIG. 43 and FIG. 44 ) show that as theH/C layer area becomes larger, the heating time increases, but thecooling time decreases. For Type-1 RHC card, the HC layer has no directphysical contact with the mechanical support to the card (e.g., sampleholder), hence the decrease in cooling cycle time is primarily due tothe increase of the thermal radiation cooling of the HC layer caused bythe increase of the HC layer's radiative cooling area.

Experiment 4 0.6 s Heating, 0.75 s Cooling Achieved with 500 nm Au H/CLayer and 500 mW Heating

In this experiment, the heating and cooling cycle of a RHC card (thesame as the one shown in FIG. 39A) with a water-like sample of 30 μmthickness and 5 μL and a 500 mW LED power were studied. The experimentaldata shown in FIG. 42 shows 10 times cycling between 65° C. to 93° C.with heating time of ˜0.65 second (an average temperature raisingramping 43° C./sec); and a cooling time of ˜0.75 sec (an averagetemperature dropping ramping 37° C./sec).

Experiment 5 H/C Layer Thickness Effects on Heating and Cooling Time

In one experiment, the effects of H/C gold layer thickness on heatingand cooling time were studied.

The example RHC card has a first plate of 100 um thick PMMA plate, asecond plate of 50 um thick PET, 30 um thick spacers array to controlsample thickness, and an H/C layer of gold is on the outer surface ofthe second plate. The first plate, the second plate, and the gold HClayer have the same area of 20 mm×20 mm. The LED heating power projectedon a ˜5 mm×5 mm heating zone of the H/C layer is 300 mW. 5 uL water-likesample on the RHC card has 30 um thickness and ˜167 mm{circumflex over( )}2 area, which is much larger than the heating zone area. The thermalcycling is between 65° C. and 95° C.

The experimental data shown in FIGS. 45A and 45B show that as the goldthickness of the HC layer changes from 300 nm to 700 nm, the heatingtime in a thermal cycle increases slightly (from 1.75 sec to 1.90 sec),but the cooling time in a thermal cycle decrease with the gold thickness(from 1.5 sec to 1.3 sec).

The cooling cycle time is shorter with the gold thickness. It suggeststhat (a) the gold HC layer thermal radiative cooling is important in thecooling of the sample and (b) the thermal radiative cooling involves athermal conduction of the heat from the sample through the gold to thegold surface for radiation. A thicker gold, a better thermal conductionof the heat from the sample to the gold HC layer edge.

The heating cycle time gets longer with an increase of the goldthickness. Clearly, a thicker gold will increase the total heatingenergy. But in this experiment, the LED heats only ˜5 mm×5 mm relevantsample area and the gold HC layer area to the maximum cyclingtemperature, and the gold thermal mass is small (due to the thinthickness), hence the increase in the total heating energy is small,leading to a weak increase of the heating cycle with the increase ofgold thickness.

Experiment 6 Effects of Heating/Cooling Layer to Sample Distance onHeating and Cooling Time

In this experiment, the effects of the distance between the HC layer andsample on heating and cooling time were studied.

The example RHC card has a first plate of 100 um thick PMMA plate, asecond plate of PET film that has different thickness for a differentRHC card, 30 um thick sample thickness controlled by spacers, and a HClayer is made of a bare 0.5 um thick gold and is on the outer surface ofthe second plate. The first plate, the second plate, and the gold HClayer have the same area of 20 mm×20 mm. The LED heating power projectedon ˜5 mm×5 mm heating zone of the H/C layer is 300 mW. 5 uL water-likesample on the RHC card has 30 um thickness and ˜167 mm² area, which ismuch larger than the heating zone area. The thermal cycling is between65° C. and 95° C.

The distance between the HC layer and sample is the distance between thegold surface that is in contact with a second plate surface and thesample surface that is in contact with another second plate surface(i.e., the gold to the sample distance).

The experimental data shown in FIGS. 46B and 46C show that as thethickness of the second plate changes (hence the gold to the sampledistance) from 25 um to 1000 um, both the heating cycle time and thecooling cycle time increase; however, the heating cycle time increaseswith the second plate thickness far more significantly than the coolingcycle time.

The data suggests that as the increase in the second plate thicknesswould cause significant increase in the energy required for heating andcooling the second plate, and significant reduction of thermalconduction between the sample and the HC layer.

For fast heating and cooling, one should reduce the thickness of thesecond plate (which is physically sandwiched by the sample and the HClayer), which should be as thin as possible. A preferred thickness ofthe second plate is 25 nm or less. Another preferred thickness of thesecond plate is 10 nm or less.

Experiment 7 Sample Thickness Effects on Heating and Cooling Time

In this experiment, the effects of the thickness of the samplesandwiched between two plates on heating and cooling time were studied.

The example RHC card has a first plate of 100 um thick PMMA plate, asecond plate of 25 um thick PET film, a periodic array of spacers tocontrol the sample thickness, and a HC layer is made of a bare 0.5 umthick gold and is on the outer surface of the second plate. A water-likesample has a different gap (i.e. thickness) for each different RHC card.The first plate, the second plate, and the gold HC layer have the samearea of 20 mm×20 mm. The LED heating power projected on ˜5 mm×5 mmheating zone of the H/C layer is 300 mW. Water-like sample on the RHCcard has ˜167 mm² area, which is much larger than the heating zone area.The thermal cycling is between 65° C. and 95° C.

The experimental data shown in FIGS. 48A and 48B show that as the samplethickness changes from 10 um to 100 um, both the heating cycle time andthe cooling cycle time increase, however the heating cycle timeincreases with the second plate thickness far more significantly thanthe cooling cycle time.

The data suggests that the increase in the sample thickness would causesignificant increase in the energy required for heating and cooling thesample.

For fast heating and cooling, one should reduce the sample thicknessshould be as thin as possible. A preferred thickness of the sample is 30um or less. Another preferred thickness of the sample is 10 um or less.Another preferred thickness of the sample is 5 um or less.

Experiment 8 LED Power Effects on Heating and Cooling Time

In this experiment, the effects of LED power on heating and cooling timewere studied. The example RHC card has a first plate of 50 um thick PMMAplate, a second plate of 25 um thick PET, a HC layer is on the outersurface of the second plate. The first plate, the second plate, and thegold/black-paint HC layer have the same area of 20 mm×20 mm. The firstplate has, on its inner surface, a periodic array of spacers that has a30 um height, a 30 um×40 um lateral sectional size and an 80 um interspacer distance. The HC layer comprise an Au (gold) film of 500 nm thickand a black paint layer. The gold film has one surface in contact withthe second plate outer surface, and another surface being painted with ablack paint. The black paint is a commercial product of a filmcomposited of black carbon nanoparticle and polymer mixture. The blackpaint had an average thickness of ˜9 um (˜2 um thickness variation).

The heating power, provided by a blue (450 nm peak wavelength) LED, wasprojected on ˜5 mm×5 mm heating zone of the H/C layer, and the power wasvaried from 100 mw to 500 mW. The sample liquid is 5 uL temperaturesensitive dye LDS698 2 mg/mL in 60% water and 40% DMSO. The temperaturesensitive dye allows us to measure the sample temperature optically. The5 uL sample on the RHC card has 30 um thickness and ˜167 mm² area, whichis much larger than the heating zone area.

Experimental data shown in FIG. 48A shows the relationship between theheating time and the heating source power, illustrating experimentaldata of the time needed for heating from 65° C. to 93° C. with heatingLED power strength from 100 mW to 500 mW on the RHC card.

Experimental data shown in FIG. 48B shows the relationship between thecooling time and the heating source power, illustrating the time neededfor cooling from 93° C. to 65° C. The heating/cooling time results arealso shown in Table 1.

The experimental data show that for the given sample holder (i.e. RHCcard) as the LED power was increased from 100 mW to 500 mW, the heatcycle time drops from 14 sec to 0.4 sec, while the cooling cycle time isnearly constant.

The experimental data suggest that for a low heating power (i.e. lowheating power density), it would take a longer time to deliver a fixedamount of energy, and the longer time will increase the energy loss inthermal conduction and radiation loss, and hence increase wasted energy.For cooling, since the amount of the thermal energy stored in a fixedvolume sample at a given temperature is fixed, regardless how long toreach that temperature, the cooling time is almost independent of theheating cycle time.

The experiment showed that in order to reduce the total thermal cycletime, one should increase the heating power.

TABLE 1 LED power effects on RHC heating and cooling time Heating LEDpower 100 200 300 400 500 mW mW mW mW mW Heating time 14 1.76 0.87 0.530.4 (65-93° C.) (second) Cooling time 0.75 0.77 0.8 0.7 0.8 (65-93° C.)(second) Heating time + 14.75 2.53 1.67 1.23 1.2 Cooling time (second)

Experiment 9 H/C Layer Materials Effects on Heating and Cooling Time

In this experiment, the effects of H/C layer materials on heating andcooling time were studied. The example RHC card has a first plate of 50um thick PMMA plate, a second plate of 25 um thick PET film, a periodicarray of spacers to regulate a water-like sample to a thickness to 30 umthickness, and a HC layer is on the outer surface of the second plate.The HC layer has a different material for each different RHC card. Thefirst plate, the second plate, and the HC layer have the same area of 20mm×20 mm. The LED heating power projected on ˜5 mm×5 mm heating zone ofthe H/C layer is 300 mW. 5 uL water-like sample on the RHC card has 30um thickness and ˜167 mm² area, which is much larger than the heatingzone area. The thermal cycling is between 65° C. and 93° C.

The experimental data shown in FIGS. 49A and 49B show that for the threedifferent HC layer materials tested, the heating cycle time and coolingcycle time is 0.75 sec and 0.75 sec, respectively for sample holder withthe HC layer of Au (500 nm thick) plus 9 um black paint, 1 sec and 1.1sec for the sample holder with the HC layer of Au (500 nm thick) only,and 1.75 sec and 1 sec for the sample holder with the HC layer of A1(500 nm thick) plus 9 um black paint.

This experiment indicates the significance of a good lateral thermalconductance in thermal radiation cooling. Compared with the gold with ablack paint, the aluminum plus black paint has nearly the sample lightabsorption (hence radiation), but much poorer lateral thermalconduction, which makes the effect thermal radiation area much less,because the heat cannot spread laterally as much.

The experiments show that a preferred embodiment for the material for HClayer is a thin gold film plus a black paint.

Experiment 10 Demonstration of 0.73 sec Thermal Cycling Time (0.23 secHeating Time and 0.5 sec Cooling Time sec)

In this experiment, for a thermal cycling between 65 C to 93 C, a RHCcard (Card-B) experimentally demonstrated 0.73 sec thermal cycling time(0.23 sec heating time and 0.5 sec cooling time) and another RHC card(Card-A) experimentally demonstrated 0.9 sec thermal cycling time (0.3sec heating time and 0.6 sec cooling time)

FIG. 50A illustrates a sample holder having two plates, each of thembeing a high-density polyethylene (HDPE) film that has about a 10 μmthickness, about 20 mm wide and about 20 mm long, according to someembodiments. The spacers that control the sample thickness were about 24μm diameter soda lime spheres with concentration of approximately 60mg/mL. The sphere spacers were mixed with the sample.

FIG. 50B illustrates a sample holder having a first plate of poly(methylmethacrylate) (PMMA) film of 25 um thickness, and a second plate ofhigh-density polyethylene (HDPE) film of 10 um thickness. Both plateshave the same area of 20 mm×20 mm. The first plate has, on its innersurface, a periodic array of spacers of 10 um height, 30 um×40 um sizeand 80 um inter spacer distance.

Both sample holder embodiments illustrated in FIGS. 50A and 50B have aH/C layer on the entire outer surface of the second plate. The H/C layercomprises an Au film with 500 nm thickness, that has one surface incontact with the second plate outer surface, and another surface beingpainted with a black paint. The black paint is a commercial product of afilm composed a black carbon nanoparticle and polymer mixture, and thefilm painted has average thickness of 9 um and 2 um thickness variation.

The sample is a liquid temperature sensitive dye LDS698 with 2 mg/mLconcentration in 60% water and 40% DMSO. The volume of the sample is 5uL for the sample holder in FIG. 50A and 3 uL for the sample holder inFIG. 50B.

The heating source, which is a blue light emitting diode (LED) with acentral wavelength of 450 nm, projected 500 mW energy on the black paintlayer of the HC layer, forming a heating zone of an ˜5 mm×5 mm area inthe center of the second plate.

The experimental data (Table 2) show that for a thermal cycling of 65°C. to 93° C., the sample holder of FIG. 50A (has 0.3 s heating cycletime and 0.60 s cooling time, hence 0.90 sec total thermal cycle time;and the sample holder of FIG. 50B has 0.23 s heating cycle time and 0.50s cooling time, hence 0.73 sec total thermal cycle time.

TABLE 2.1 RHC parameters RHC card setup Card 1^(st) Plate 2^(nd) PlateSpacer FIG. 10 um 10 um HDPE 24 um 50A HDPE diameter beads FIG. 24 um 10um HDPE 10 um 50B PMMA height pillar

TABLE 2.2 Heating/Cooling performance Heating/Cooling performanceHeating time (s) Cooling time (s) Cycling Card 65° C. to 93° C. 93° C.to 65° C. time (s) FIG. 50A 0.30 s 0.60 s 0.90 s FIG. 50B 0.23 s 0.50 s0.73 s

Experiment 11 Effects of Using Sample Support and Sample Adaptor onHeating and Cooling Time

In this experiment, the effects of using sample holder support andsample adaptor on heating and cooling time were studied.

In the experiment, a RHC card was put on a mechanical sample cardsupport (termed “card support”), and then the card support was slideinto an adaptor. The thermal cycling time was measured for the cases of(a) the RHC card only, (b) the RHC card on the card support, and (c) theRHC card on the card support and the card support is slid into theadaptor.

In some embodiments, a sample holder (illustrated in FIGS. 51A and 51B)comprises a first plate is a poly(methyl methacrylate) (PMMA) film thathas a 10 um to 50 um thickness, a 22 mm width and a 27 mm length. Thefirst plate has, on its inner surface, a periodic array of spacershaving a 30 um height, a 30 um×40 um sectional size and 80 um interspacer distance.

In some embodiments, the second plate is a polyethylene terephthalate(PET) film or high-density polyethylene film that has a 10 um to 50 umthickness, a 20 mm width and a 27 mm length. The heating/cooling layercovers the entire outer surface of the second plate. The heating/coolinglayer comprises an Au film with 100 nm to 500 nm thickness that has onesurface in contact with the second plate outer surface, and anothersurface being painted with a black paint. The black paint has an averagethickness of 9 um.

According to some embodiments, a card support (as illustrated in FIGS.51A and 51B) comprises 1 mm thick PMMA plate of 24 mm width and 32 mmlength that has a 15 mm×15 mm square hole in the center. The RHC cardand the card support were glued together using 10-15 um thick adhesivebetween the black paint of the RHC card and a surface of the cardsupport as illustrated in FIG. 51B.

According to some embodiments, the card adapter comprises an assembly oftwo U shaped frames that a sample card can slide in or out of the U. Oneof U shape frame is made of a plastic and the other piece is made of analuminum, where the two U shape frame are assembled in parallel with agap between them, and the gap is the slot for the sample card to slide.An example of the card adaptor is to cut a conventional SD cardconnecter into U shape (cut from the back end).

In testing the thermal cycling time of a RHC card of 50 nm thick firstcard and 25 nm thick PET second card (both has 20 mm×20 mm area), thesample liquid was 5 uL temperature sensitive dye LDS698 2 mg/mL in 60%water and 40% DMSO, and a blue 450 nm LED) was projected on the blackpaint with 5 mm×5 mm area (forming the heating zone) and 300 mW power.

The experimental data shown in FIG. 52 demonstrates that for a thermalcycling between 65° C. and 93° C., (a) for just the RHC card only, theheating cycle time was 0.67 sec, the cooling cycle was 0.9 sec, andtotal thermal cycle time was 1.57 sec; (b) for the RHC card on the cardsupport, the heating cycle time was 0.77 sec, the cooling cycle was 0.87sec, and total thermal cycle time was 1.64 sec; and (c) for the RHC cardon the card support and the card support is slide into the adaptor, theheating cycle time was 0.93 sec, the cooling cycle was 0.7 sec, andtotal thermal cycle time was 1.63 sec.

The experimental data suggest that by making a RHC card coolingprimarily based on thermal radiative cooling, the RHC card can besupported by a card support and the card support can be inserted into anadaptor while increasing the thermal cycle time less than 4%.

Experiment 12 Effects of Using Extra Heat Sink

In this experiment, the effects of adding an external heat sink onheating and cooling time were studied. In this experiment, rather thanlet heat from the RHC card radiate into the environment, a Peltier coolwas put in touch with an edge of the HC layer of a RHC card.

The RHC card has a first plate of 50 um thick PMMA plate, a second plateof 50 um thick PET film, a periodic array of spacers to control thesample thickness to 30 um, and a HC layer is made of a bare 0.3 um thickgold and is on the outer surface of the second plate. The first platehas an area of 20 mm×20 mm. The second plate and the gold HC layer havethe same area of 30 mm×30 mm.

The LED heating power projected on ˜5 mm×5 mm heating zone of the H/Clayer is 500 mW. 5 uL water-like sample on the RHC card has 30 umthickness and ˜167 mm² area, which is much larger than the heating zonearea. The thermal cycling is between 65° C. and 93° C.

In some setups, a Peltier cooler, providing 0 C heat sink, is either incontact with or nearby the HC layer by overlapping 3 mm edge with thesecond plate. In a reference setup, there was no Peltier cooler. Sampleliquid is 5 uL temperature sensitive dye LDS698 with 2 mg/mLconcentration in 60% water and 40% DMSO.

The experimental data (Table 3) show that without the Peltier cooler,the liquid inside RHC card's heating time from 65° C. to 93° C. is 0.63s, while the cooling time from 93° C. to 65° C. is 1.2 s, and that withPeltier cooler in contact with the Au film, the liquid inside RHC card'sheating time from 65° C. to 93° C. is increased to 0.73 s, while thecooling time from 93° C. to 65° C. is shorten to 0.93 s. Using thePeltier cooler, the total thermal cycle time is reduced from 1.83 to1.66. This was achieved by a slightly increase of the heating cycletime, but a significantly reduce of the cooling cycle time.

TABLE 3 Effects of Using Extra Heat Sink with RHC card No Peltier on RHCSetup Peltier edge; touching Au Heating Time 0.63 0.73 (65-93° C.)(seconds) Cooling Time 1.2 0.93 (93-65° C.) (seconds) Heating Time +1.83 1.66 Cooling Time (seconds)

Sample Card (i.e. RHC Card)

Certain exemplary embodiments of the key components of a sample card(i.e. RHC card) are given below.

Sample Thickness

To reduce the thermal mass of the sample as well as reduce the thermalconvention loss in the sample, in some embodiments, the average samplethickness at the region being heated by the heating/cooling layer is 500um or less, 200 um or less, 100 um or less, 50 um or less, 20 um orless, 10 um or less, 5 um or less, 2 um or less, 1 um or less, 500 nm orless, 300 nm or less, 100 nm or less, 50 nm or less, or a range betweenany two of the values.

One preferred average sample thickness at the region being heated by theheating/cooling layer is from 0.1 um to 0.5 um, 0.5 um to 10 um, 10 umto 20 um, 20 um to 30 um, 30 um to 50 um, from 50 um to 80 um, 80 um to100 um, or 100 um to 150 um.

Experiment 13 Example of Real Time PCR Amplification with RHC Card andSystem

The RHC card in this experiment has a first plate of 50 um thick PMMAplate, a second plate of 25 um thick PET, a H/C layer is on the outersurface of the second plate. The gold/black-paint HC layer have an areaof 10 mm in diameter. The first plate has, on its inner surface, aperiodic array of spacers. The HC layer comprise a thin Au (gold) filmand a black paint layer. The gold film has one surface in contact withthe second plate outer surface, and another surface in contact with ablack paint. The black paint is a commercial product of a filmcomposited of black carbon nanoparticle and polymer mixture. The blackpaint had an average thickness of ˜9 um (˜2 um thickness variation).

PCR (real-time PCR) reagents for amplification of Staphylococcus aureusgenomic DNA with a total volume of 20 uL comprise MSSA Forward primer,MSSA Reverse primer and Cy5 labeled DNA probe with AptaTaq DNA buffer,Aptataq Polymerase, MgCl₂, dNTP, bovine serum albumin (BSA), templateDNA and ddH₂O.

In a real time PCR experiments, two positive RHC cards have shown clearincrease in fluorescence signal vs. cycle number, especially after 20cycles' amplification. While one negative RHC card does not have obviousincreased fluorescence signal vs. cycle number. After 40 cycles ofamplification in RHC system, the PCR products in RHC cards are extractedand a nucleic acid gel run is performed to confirm that only twopositive RHC cards have successful amplification band.

Sample Wells

In certain embodiments, one or both of the plates have sample wells,wherein the well regulates the maximum volume of the sample in the welland prevents the sample to flow into other location of the plates.

Plate Thickness

To reduce the thermal mass of the first and second plates as well asreduce the lateral thermal conduction loss in the plates, the thicknessof the first plate and the second plate is preferred to be thin.

In certain embodiments, the first plate or the second plate has athickness of 2 nm or less, 10 nm or less, 100 nm or less, 200 nm orless, 500 nm or less, 1000 nm or less, 2 μm (micron) or less, 5 μm orless, 10 μm or less, 20 μm or less, 50 μm or less, 100 μm or less, 150μm or less, 200 μm or less, 300 μm or less, 500 μm or less, 800 μm orless, 1 mm (millimeter) or less, 2 mm or less, 3 mm or less, 5 mm orless, 10 mm or less, or in a range between any two of these values.

In some embodiments, the first plate or the second plate has a thicknessof 10 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 1 um, 2.5 um, 5 um, 10um, 25 um, 50 um, 100 um, 200 um, or 500 um, 1000 um, or in a rangebetween any of the two values.

The first plate and the second plate can have the same thickness or adifferent thickness, and can be made of the same materials or differentmaterials.

In some preferred embodiments, the first plate or the second plate has athickness in a range of between 10 nm and 500 nm, 500 nm and 1 um, 1 umand 2.5 um, 2.5 um and 5 um, 5 um and 10 um, 10 um and 25 um, 25 um and50 um, 50 um and 100 um, 100 um and 200 um, or 200 um and 500 um, or 500um and 1000 um.

A preferred thickness of the first plate or the second plate is 10 nm orless, 100 nm or less, 200 nm or less, 500 nm or less, 1000 nm or less, 2μm (micron) or less, 5 μm or less, 10 μm or less, 20 μm or less, 50 μmor less, 100 μm or less, 150 μm or less, 200 μm or less, 300 μm or less,500 μm or less, or in a range between any two of the values.

In some preferred embodiments, the thickness of the plate that has theheating/cooling layer is thinner than the other plate that does not havea heater.

In some preferred embodiments, the first plate has a thickness of 100nm, 200 nm, 500 nm, 1 μm (micron), 2 μm, 5 μm, 10 μm, 25 μm, 50 μm, 100μm, 125 μm, 150 μm, 175 μm, 200 μm, 250 μm, or in a range between anytwo of the values; while the second plate has a thickness of 25 μm, 50μm, 100 μm, 125 μm, 150 μm, 175 μm, 200 μm, 250 μm, 500 μm, 1 mm, 1.5mm, 2 mm, or in a range between any two of the values,

In some embodiments, the average thickness for at least one of theplates is in the range of 1 to 1000 μm, 10 to 900 μm, 20 to 800 μm, 25to 700 μm, 25 to 800 μm, 25 to 600 μm, 25 to 500 μm, 25 to 400 μm, 25 to300 μm, 25 to 200 μm, 30 to 200 μm, 35 to 200 μm, to 200 μm, 45 to 200μm, or 50 to 200 μm.

In some embodiments, the average thickness for at least one of theplates is in the range of 50 to 75 μm, 75 to 100 μm, 100 to 125 μm, 125to 150 μm, 150 to 175 μm, or 175 to 200 μm.

In some embodiments, the average thickness for at least one of theplates is about 50 μm, about 75 μm, about 100 μm, about 125 μm, about150 μm, about 175 μm, or about 200 μm.

Plate Area. In some embodiments, the first plate and/or the second platehas a lateral area of 1 mm² (square millimeter) or less, 10 mm² or less,25 mm² or less, 50 mm² or less, 75 mm² or less, 1 cm² (squarecentimeter) or less, 2 cm² or less, 3 cm² or less, 4 cm² or less, 5 cm²or less, 10 cm² or less, 20 cm² or less, 30 cm² or less, 50 cm² or less,100 cm² or less, 500 cm² or less, 1,000 cm² or less, 5,000 cm² or less,10,000 cm² or less, or in a range between any two of these values.

In preferred embodiments, the first plate and/or the second plate has alateral area in a range of 1 mm² (square millimeter) to 10 mm², 10 mm²to 50 mm², 50 mm² to 100 mm², 1 cm² to 5 cm², 5 cm² to 20 cm², 20 cm² to50 cm², 50 cm² to 100 cm², 100 cm² to 500 cm², 500 cm² to 1000 cm², or1000 cm² to 10,000 cm².

In some embodiments, the first plate and the second plate have the samelateral dimension. In some embodiments, one of the plates has an areathat is different from the other plates by 10% or less, 30% or less, 50%or less, 80% or less, 90% or less, 95% or less, 99% or less, or in arange between any two of these values (take the largest plate is thebase in calculation the different percentage).

In some embodiment, the first plate and/or the second plate has a widthor a length of 5 mm, 10 mm, 20 mm, 25 mm, 30 mm, 40 mm, 50 mm, 75 mm,100 mm, or in a range between any two of these values.

In preferred embodiments, the first plate and/or the second plate has awidth or a length in a range of 5 mm to 10 mm, 20 mm to 30 mm, 30 mm to50 mm, 50 mm to 75 mm, or 75 mm to 100 mm.

In one preferred embodiment, the plate has a width or length in a rangeof 5 mm to, 50 mm. In another preferred embodiment, the plate has awidth in a range of 5 mm to 50 mm and a length in a range of 6 mm to 70mm.

Materials for Plates

In some embodiments, the materials for the first plate and the secondplates, contain but are not limit to polymers (e.g. plastics) oramorphous organic materials. The polymer materials include, not limitedto, acrylate polymers, vinyl polymers, olefin polymers, cellulosicpolymers, noncellulosic polymers, polyester polymers, Nylon, cyclicolefin copolymer (COC), poly(methyl methacrylate) (PMMA), polycarbonate(PC), cyclic olefin polymer (COP), liquid crystalline polymer (LCP),polyimide (PA), polyethylene (PE), polyimide (PI), polypropylene (PP),poly(phenylene ether) (PPE), polystyrene (PS), polyoxymethylene (POM),polyether ether ketone (PEEK), polyether sulfone (PES), poly(ethylenephthalate) (PET), polytetrafluoroethylene (PTFE), polyvinyl chloride(PVC), polyvinylidene fluoride (PVDF), polybutylene terephthalate (PBT),fluorinated ethylene propylene (FEP), perfluoroalkoxyalkane (PFA),polydimethylsiloxane (PDMS), rubbers, or any combinations of thereof.

In some embodiments, the materials for the first plate and the secondplate contain but are not limit to inorganic materials includingdielectric materials of silicon oxide, porcelain, orcelain (ceramic),mica, glass, oxides of various metals, etc.

In some embodiments, the materials for the first plate and the secondplate contain but are not limit to inorganic materials includingaluminum oxide, aluminum chloride, cadmium sulfide, gallium nitride,gold chloride, indium arsenide, lithium borohydride, silver bromide,sodium chloride, graphite, carbon nanotubes, carbon fibers, etc.

In some embodiments, the materials for the first plate and the secondplate contain but are not limit to metals (e.g. gold, copper, aluminum,etc.) and alloys.

In some embodiments, the materials for the first plate and the secondplate are made of multi-layers and/or mixture of the materials listedabove.

Heating Layer and Cooling Layer

In certain embodiments, a heating layer (112-1) and a cooling layer(112-2) comprises high K material and/or a high KC ratio material. Thehigh K and/or high KC ratio material comprises materials/structures,such as, but not limited to, metallic film, semiconductors, semimetals,plasmonic surface, metamaterials (e.g. nanostructures), black silicon,graphite, carbon nanotube, silicon sandwich, graphene, superlattice,plasmonic materials, any material/structure that is capable ofefficiently absorbing the electromagnetic wave and converting theabsorbed energy into thermal energy, and any combination thereof.

For a heating layer that is heated by an optical heating source, aheating layer comprises a material layer that significantly absorb theradiated energy from the optical heating source. The significantabsorption means that the heating/cooling layer absorbs the radiatedenergy from the optical heating source more significantly than thesample and the plates.

In certain embodiments, the heating/cooling layer has thickness in therange of 50 nm to 15 um. In certain embodiments, the heating/coolinglayer comprise a high K layer that has thickness in the range of 100 nmto 1 um.

In some embodiments, the dimension of the light heating area is about 1um, 2 um, 5 um, 10 um, 20 um, 50 um, 100 um, 200 um, 500 um, 1 mm, 2 mm,5 mm, 10 mm, 20 mm, 50 mm, or 100 mm, or in a range between any of thetwo values. In various embodiments, the size and shape of the lightheating areas can vary.

In some embodiments, the heating/cooling layer comprise adot-coupled-dots-on-pillar antenna (D2PA) array, such as, but notlimited to the D2PA array described in U.S. Provisional PatentApplication No. 61/347,178, which was filed on May 21, 2010, U.S.Provisional Patent Application 61/622,226, which was filed on Apr. 10,2012, U.S. PCT Application No. PCT/US2011/037455, which was filed on May20, 2011, PCT Application No. PCT/US2013/032347, which was filed on Mar.15, 2013, and U.S. patent application Ser. No. 13/699,270, which wasfiled on Jun. 13, 2013, the complete disclosures of which are herebyincorporated by reference for all purposes.

In some embodiments, there can be more than one heating/cooling layer.For examples, at least two surfaces of any of the first or second plateshave a heating/cooling layer.

In some embodiments, the heating/cooling layer can be two-layermaterials: one layer for heating and one for cooling, and the two-layermaterials can be on the same surface of any of the first or secondplate. For sample, the heating layer can be on the outer surface of thesecond plate, while the cooling layer is on the outer surface or theinner surface of the first plate. Even the cooling layer is on the outersurface of the first plate, which should be efficient in cooling thesample as long as the first plate has thin thickness (e.g., 25 um orless).

Spacers

In some embodiments of the present invention there are spacers betweenthe two plates. In some embodiments, at least one of the spacers is inthe sample contact area. In some embodiments, the spacers have uniformheight. In some embodiments, the thickness of the sample is the sampleas the height of the spacers. In some embodiments, the spacers are fixedon one of the plates.

Spacers' Function. In present invention, the spacers are configured tohave one or any combinations of the following functions and properties:the spacers are configured to (1) control, together with the plates, thethickness of the sample or a relevant volume of the sample (Preferably,the thickness control is precise, or uniform or both, over a relevantarea); (2) allow the sample to have a compressed regulated open flow(CROF) on plate surface; (3) not take significant surface area (volume)in a given sample area (volume); (4) reduce or increase the effect ofsedimentation of particles or analytes in the sample; (5) change and/orcontrol the wetting propertied of the inner surface of the plates; (6)identify a location of the plate, a scale of size, and/or theinformation related to a plate, or (7) do any combination of the above.

Spacer architectures and shapes. To achieve desired sample thicknessreduction and control, in certain embodiments, the spacers are fixed onits respective plate. In general, the spacer can have any shape, as longas the spacers are capable of regulating the sample thickness during aCROF process, but certain shapes are preferred to achieve certainfunctions, such as better uniformity, less overshoot in pressing, etc.

The spacer(s) is a single spacer or a plurality of spacers. (e.g. anarray). Some embodiments of a plurality of spacers is an array ofspacers (e.g. pillars), where the inter-spacer distance is periodic oraperiodic, or is periodic or aperiodic in certain areas of the plates,or has different distances in different areas of the plates.

There are two kinds of the spacers: open-spacers and enclosed-spacers.The open-spacer is the spacer that allows a sample to flow through thespacer (i.e. the sample flows around and pass the spacer. For example, apost as the spacer), and the enclosed spacer is the spacer that stop thesample flow (i.e. the sample cannot flow beyond the spacer. For example,a ring shape spacer and the sample is inside the ring). Both types ofspacers use their height to regular the final sample thickness at aclosed configuration.

In some embodiments, the spacers are open-spacers only. In someembodiments, the spacers are enclosed-spacers only. In some embodiments,the spacers are a combination of open-spacers and enclosed-spacers.

The term “pillar spacer” means that the spacer has a pillar shape andthe pillar shape refers to an object that has height and a lateral shapethat allow a sample to flow around it during a compressed open flow. Insome embodiments, the spacers have a flat top (e.g. pillars with a flattop to contact a plate).

In some embodiments, the lateral shapes of the pillar spacers are theshape selected from the groups of (i) round, elliptical, rectangles,triangles, polygons, ring-shaped, star-shaped, letter-shaped (e.g.L-shaped, C-shaped, the letters from A to Z), number shaped (e.g. theshapes like 0 1, 2, 3, 4, . . . to 9); (ii) the shapes in group (i) withat least one rounded corners; (iii) the shape from group (i) withzig-zag or rough edges; and (iv) any superposition of (i), (ii) and(iii). For multiple spacers, different spacers can have differentlateral shape and size and different distance from the neighboringspacers.

In some embodiments, the spacers can be and/or can include posts,columns, beads, spheres, and/or other suitable geometries. The lateralshape and dimension (i.e., transverse to the respective plate surface)of the spacers can be anything, except, in some embodiments, thefollowing restrictions: (i) the spacer geometry will not cause asignificant error in measuring the sample thickness and volume; or (ii)the spacer geometry would not prevent the out-flowing of the samplebetween the plates (i.e. it is not in enclosed form). But in someembodiments, they require some spacers to be closed spacers to restrictthe sample flow.

In some embodiments, the shapes of the spacers have rounded corners. Forexample, a rectangle shaped spacer has one, several or all cornersrounded (like a circle rather 90 degree angle). A round corner oftenmake a fabrication of the spacer easier, and in some cases less damageto a biological material.

The sidewall of the pillars can be straight, curved, sloped, ordifferent shaped in different section of the sidewall. In someembodiments, the spacers are pillars of various lateral shapes,sidewalls, and pillar-height to pillar lateral area ratio.

In a preferred embodiment, the spacers have shapes of pillars forallowing open flow.

Spacers' materials. In the present invention, the spacers are generallymade of any material that is capable of being used to regulate, togetherwith the two plates, the thickness of a relevant volume of the sample.In some embodiments, the materials for the spacers are different fromthat for the plates. In some embodiments, the materials for the spacesare at least the same as a part of the materials for at least one plate.

The spacers are made a single material, composite materials, multiplematerials, multilayer of materials, alloys, or a combination thereof.Each of the materials for the spacers is an inorganic material, amorganic material, or a mix, wherein examples of the materials are givenin paragraphs of Mat-1 and Mat-2. In a preferred embodiment, the spacersare made in the same material as a plate used in CROF.

Spacer's mechanical strength and flexibility. In some embodiments, themechanical strength of the spacers are strong enough, so that during thecompression and at the closed configuration of the plates, the height ofthe spacers is the same or significantly same as that when the platesare in an open configuration. In some embodiments, the differences ofthe spacers between the open configuration and the closed configurationcan be characterized and predetermined.

The material for the spacers is rigid, flexible or any flexibilitybetween the two. The rigid is relative to a give pressing forces used inbringing the plates into the closed configuration: if the space does notdeform greater than 1% in its height under the pressing force, thespacer material is regarded as rigid, otherwise a flexible. When aspacer is made of material flexible, the final sample thickness at aclosed configuration still can be predetermined from the pressing forceand the mechanical property of the spacer.

Spacer inside Sample. To achieve desired sample thickness reduction andcontrol, particularly to achieve a good sample thickness uniformity, incertain embodiments, the spacers are placed inside the sample, or therelevant volume of the sample. In some embodiments, there are one ormore spacers inside the sample or the relevant volume of the sample,with a proper inter spacer distance. In certain embodiments, at leastone of the spacers is inside the sample, at least two of the spacersinside the sample or the relevant volume of the sample, or at least of“n” spacers inside the sample or the relevant volume of the sample,where “n” can be determined by a sample thickness uniformity or arequired sample flow property during a CROF.

Spacer height. In some embodiments, all spacers have the samepre-determined height. In some embodiments, spacers have differentpre-determined height. In some embodiments, spacers can be divided intogroups or regions, wherein each group or region has its own spacerheight. And in certain embodiments, the predetermined height of thespacers is an average height of the spacers. In some embodiments, thespacers have approximately the same height. In some embodiments, apercentage of number of the spacers have the same height. In someembodiments, on the same plate, the spacer height in one ration isdifferent from the spacer height in another region. In some cases, theplate with different spacer height in different regions have advantagesof assaying.

The height of the spacers is selected by a desired regulated finalsample thickness and the residue sample thickness. The spacer height(the predetermined spacer height) and/or sample thickness is 3 nm orless, 10 nm or less, 50 nm or less, 100 nm or less, 200 nm or less, 500nm or less, 800 nm or less, 1000 nm or less, 1 um or less, 2 um or less,3 um or less, 5 um or less, 10 um or less, 20 um or less, 30 um or less,50 um or less, 100 um or less, 150 um or less, 200 um or less, 300 um orless, 500 um or less, 800 um or less, 1 mm or less, 2 mm or less, 4 mmor less, or a range between any two of the values.

The spacer height and/or sample thickness is between 1 nm to 100 nm inone preferred embodiment, 100 nm to 500 nm in another preferredembodiment, 500 nm to 1,000 nm in a separate preferred embodiment, 1 um(i.e., 1,000 nm) to 2 um in another preferred embodiment, 2 um to 3 umin a separate preferred embodiment, 3 um to 5 um in another preferredembodiment, 5 um to 10 um in a separate preferred embodiment, and 10 umto 50 um in another preferred embodiment, 50 um to 100 um in a separatepreferred embodiment.

In some embodiments, the spacer height and/or sample thickness is (i)equal to or slightly larger than the minimum dimension of an analyte, or(ii) equal to or slightly larger than the maximum dimension of ananalyte. The “slightly larger” means that it is about 1% to 5% largerand any number between the two values.

In some embodiments, the spacer height and/or sample thickness is largerthan the minimum dimension of an analyte (e.g. an analyte has ananisotropic shape), but less than the maximum dimension of the analyte.

For example, the red blood cell has a disk shape with a minim dimensionof 2 um (disk thickness) and a maximum dimension of 11 um (a diskdiameter). In an embodiment of the present invention, the spacers isselected to make the inner surface spacing of the plates in a relevantarea to be 2 um (equal to the minimum dimension) in one embodiment, 2.2um in another embodiment, or 3 (50% larger than the minimum dimension)in other embodiment, but less than the maximum dimension of the redblood cell. Such embodiment has certain advantages in blood cellcounting. In one embodiment, for red blood cell counting, by making theinner surface spacing at 2 or 3 um and any number between the twovalues, a undiluted whole blood sample is confined in the spacing, onaverage, each red blood cell (RBC) does not overlap with others,allowing an accurate counting of the red blood cells visually. Too manyoverlaps between the RBC's can cause serious errors in counting.

In the present invention, in some embodiments, it uses the plates andthe spacers to regulate not only a thickness of a sample, but also theorientation and/or surface density of the analytes/entity in the samplewhen the plates are at the closed configuration. When the plates are ata closed configuration, a thinner thickness of the sample gives a lessthe analytes/entity per surface area (i.e. less surface concentration).

Spacer lateral dimension. For an open-spacer, the lateral dimensions canbe characterized by its lateral dimension (sometimes being called width)in the x and y—two orthogonal directions. The lateral dimension of aspacer in each direction is the same or different.

In some embodiments, the ratio of the lateral dimensions of x to ydirection is 1, 1.5, 2, 5, 10, 100, 500, 1,000, 10,000, or a rangebetween any two of the value. In some embodiments, a different ratio isused to regulate the sample flow direction; the larger the ratio, theflow is along one direction (larger size direction).

In some embodiments, the different lateral dimensions of the spacers inx and y direction are used as (a) using the spacers as scale-markers toindicate the orientation of the plates, (b) using the spacers to createmore sample flow in a preferred direction, or both.

In a preferred embodiment, the period, width, and height.

In some embodiments, all spacers have the same shape and dimensions. Insome embodiments, each spacer has different lateral dimensions.

For enclosed-spacers, in some embodiments, the inner lateral shape andsize are selected based on the total volume of a sample to be enclosedby the enclosed spacer(s), wherein the volume size has been described inthe present disclosure; and in certain embodiments, the outer lateralshape and size are selected based on the needed strength to support thepressure of the liquid against the spacer and the compress pressure thatpresses the plates.

Aspect ratio of height to the average lateral dimension of pillarspacer. In certain embodiments, the aspect ratio of the height to theaverage lateral dimension of the pillar spacer is 100,000, 10,000,1,000, 100, 10, 1, 0.1, 0.01, 0.001, 0.0001, 0, 00001, or a rangebetween any two of the values.

Spacer height precisions. The spacer height should be controlledprecisely. The relative precision of the spacer (i.e. the ratio of thedeviation to the desired spacer height) is 0.001% or less, 0.01% orless, 0.1% or less; 0.5% or less, 1% or less, 2% or less, 5% or less, 8%or less, 10% or less, 15% or less, 20% or less, 30% or less, 40% orless, 50% or less, 60% or less, 70% or less, 80% or less, 90% or less,99.9% or less, or a range between any of the values.

Inter-spacer distance. The spacers can be a single spacer or a pluralityof spacers on the plate or in a relevant area of the sample. In someembodiments, the spacers on the plates are configured and/or arranged inan array form, and the array is a periodic, non-periodic array orperiodic in some locations of the plate while non-periodic in otherlocations.

In some embodiments, the periodic array of the spacers has a lattice ofsquare, rectangle, triangle, hexagon, polygon, or any combinations ofthereof, where a combination means that different locations of a platehas different spacer lattices.

In some embodiments, the inter-spacer distance of a spacer array isperiodic (i.e. uniform inter-spacer distance) in at least one directionof the array. In some embodiments, the inter-spacer distance isconfigured to improve the uniformity between the plate spacing at aclosed configuration.

The distance between neighboring spacers (i.e. the inter-spacerdistance) is 1 um or less, 5 um or less, 10 um or less, 20 um or less,30 um or less, 40 um or less, 50 um or less, 60 um or less, 70 um orless, 80 um or less, 90 um or less, 100 um or less, 200 um or less, 300um or less, 400 um or less, or in a range between any two of the values.

In certain embodiments, the inter-spacer distance is at 400 or less, 500or less, 1 mm or less, 2 mm or less, 3 mm or less, 5 mm or less, 7 mm orless, 10 mm or less, or any range between the values. In certainembodiments, the inter-spacer distance is a 10 mm or less, 20 mm orless, 30 mm or less, 50 mm or less, 70 mm or less, 100 mm or less, orany range between the values.

The distance between neighboring spacers (i.e. the inter-spacerdistance) is selected so that for a given properties of the plates and asample, at the closed-configuration of the plates, the sample thicknessvariation between two neighboring spacers is, in some embodiments, atmost 0.5%, 1%, 5%, 10%, 20%, 30%, 50%, 80%, or any range between thevalues; or in certain embodiments, at most 80%, 100%, 200%, 400%, or arange between any two of the values.

Clearly, for maintaining a given sample thickness variation between twoneighboring spacers, when a more flexible plate is used, a closerinter-spacer distance is needed.

In a preferred embodiment, the spacer is a periodic square array,wherein the spacer is a pillar that has a height of 2 to 4 um, anaverage lateral dimension of from 5 to 20 um, and inter-spacer spacingof 1 um to 100 um.

In a preferred embodiment, the spacer is a periodic square array,wherein the spacer is a pillar that has a height of 2 to 4 um, anaverage lateral dimension of from 5 to 20 um, and inter-spacer spacingof 100 um to 250 um.

In a preferred embodiment, the spacer is a periodic square array,wherein the spacer is a pillar that has a height of 4 to 50 um, anaverage lateral dimension of from 5 to 20 um, and inter-spacer spacingof 1 um to 100 um.

In a preferred embodiment, the spacer is a periodic square array,wherein the spacer is a pillar that has a height of 4 to 50 um, anaverage lateral dimension of from 5 to 20 um, and inter-spacer spacingof 100 um to 250 um.

The period of spacer array is between 1 nm to 100 nm in one preferredembodiment, 100 nm to 500 nm in another preferred embodiment, 500 nm to1000 nm in a separate preferred embodiment, 1 um (i.e. 1000 nm) to 2 umin another preferred embodiment, 2 um to 3 um in a separate preferredembodiment, 3 um to 5 um in another preferred embodiment, 5 um to 10 umin a separate preferred embodiment, and 10 um to 50 um in anotherpreferred embodiment, 50 um to 100 um in a separate preferredembodiment, 100 um to 175 um in a separate preferred embodiment, and 175um to 300 um in a separate preferred embodiment.

Spacer density. The spacers are arranged on the respective plates at asurface density of greater than one per um², greater than one per 10um², greater than one per 100 um², greater than one per 500 um², greaterthan one per 1,000 um², greater than one per 5,000 um², greater than oneper 0.01 mm², greater than one per 0.1 mm², greater than one per 1 mm²,greater than one per 5 mm², greater than one per 10 mm², greater thanone per 100 mm², greater than one per 1000 mm², greater than one per10000 mm², or a range between any two of the values.

The spacers are configured to not take significant surface area (volume)in a given sample area (volume);

Ratio of spacer volume to sample volume. In many embodiments, the ratioof the spacer volume (i.e., the volume of the spacer) to sample volume(i.e. the volume of the sample), and/or the ratio of the volume of thespacers that are inside of the relevant volume of the sample to therelevant volume of the sample are controlled for achieving certainadvantages. The advantages include, but not limited to, the uniformityof the sample thickness control, the uniformity of analytes, the sampleflow properties (i.e., flow speed, flow direction, etc.).

In certain embodiments, the ratio of the spacer volume r) to samplevolume, and/or the ratio of the volume of the spacers that are inside ofthe relevant volume of the sample to the relevant volume of the sampleis less than 100%, at most 99%, at most 70%, at most 50%, at most 30%,at most 10%, at most 5%, at most 3% at most 1%, at most 0.1%, at most0.01%, at most 0.001%, or a range between any of the values.

Spacers fixed to plates. The inter spacer distance and the orientationof the spacers, which play a key role in the present invention, arepreferably maintained during the process of bringing the plates from anopen configuration to the closed configuration, and/or are preferablypredetermined before the process from an open configuration to a closedconfiguration.

Some embodiments of the present invention is that the spacers are fixedon one of the plates before the plates are brought to the closedconfiguration. The term “a spacer is fixed with its respective plate”means that the spacer is attached to a plate and the attachment ismaintained during a use of the plate. An example of “a spacer is fixedwith its respective plate” is that a spacer is monolithically made ofone piece of material of the plate, and the position of the spacerrelative to the plate surface does not change. An example of “a spaceris not fixed with its respective plate” is that a spacer is glued to aplate by an adhesive, but during a use of the plate, the adhesive cannothold the spacer at its original location on the plate surface (i.e. thespacer moves away from its original position on the plate surface).

In some embodiments, at least one of the spacers are fixed to itsrespective plate. In certain embodiments, at two spacers are fixed toits respective plates. In certain embodiments, a majority of the spacersare fixed with their respective plates. In certain embodiments, all ofthe spacers are fixed with their respective plates.

In some embodiments, a spacer is fixed to a plate monolithically.

In some embodiments, the spacers are fixed to its respective plate byone or any combination of the following methods and/or configurations:attached to, bonded to, fused to, imprinted, and etched.

The term “imprinted” means that a spacer and a plate are fixedmonolithically by imprinting (i.e. embossing) a piece of a material toform the spacer on the plate surface. The material can be single layerof a material or multiple layers of the material.

The term “etched” means that a spacer and a plate are fixedmonolithically by etching a piece of a material to form the spacer onthe plate surface. The material can be single layer of a material ormultiple layers of the material.

The term “fused to” means that a spacer and a plate are fixedmonolithically by attaching a spacer and a plate together, the originalmaterials for the spacer and the plate fused into each other, and thereis clear material boundary between the two materials after the fusion.

The term “bonded to” means that a spacer and a plate are fixedmonolithically by binding a spacer and a plate by adhesion.

The term “attached to” means that a spacer and a plate are connectedtogether.

In some embodiments, the spacers and the plate are made in the samematerials. In other embodiment, the spacers and the plate are made fromdifferent materials. In other embodiment, the spacer and the plate areformed in one piece. In other embodiment, the spacer has one end fixedto its respective plate, while the end is open for accommodatingdifferent configurations of the two plates.

In other embodiment, each of the spacers independently is at least oneof attached to, bonded to, fused to, imprinted in, and etched in therespective plate. The term “independently” means that one spacer isfixed with its respective plate by a same or a different method that isselected from the methods of attached to, bonded to, fused to, imprintedin, and etched in the respective plate.

In some embodiments, at least a distance between two spacers ispredetermined (“predetermined inter-spacer distance” means that thedistance is known when a user uses the plates).

In some embodiments of all methods and devices described herein, thereare additional spacers besides to the fixed spacers.

In one preferred embodiment, the spacers are monolithically made on thePlate by embossing (e.g. nanoimprinting) a thin plastic film using amold, and are made of the same materials, and the thickness of the Plateis from 50 um to 500 um.

In one preferred embodiment, the spacers are monolithically made on thePlate by embossing (e.g. nanoimprinting) a thin plastic film using amold, and are made of the same materials, and the thickness of the Plateis from 50 um to 250 um.

In one preferred embodiment, the spacers are monolithically made on thePlate and are made of the same materials, and the thickness of the Plateis from 50 um to 500 um.

In one preferred embodiment, the spacers are monolithically made on thePlate a thin plastic film using a mold, and are made of the samematerials, and the thickness of the Plate is from 50 um to 250 um.

In one preferred embodiment, the spacers are monolithically made on thePlate by embossing (e.g. nanoimprinting) a thin plastic film using amold, and are made of the same materials, where the plastic film areeither PMMA (polymethyl methacrylate) of PS (polystyrene).

In one preferred embodiment, the spacers are monolithically made on thePlate by embossing (e.g. nanoimprinting) a thin plastic film using amold, and are made of the same materials, where the plastic film areeither PMMA (polymethyl methacrylate) of PS (polystyrene) and thethickness of the Plate is from 50 um to 500 um.

In one preferred embodiment, the spacers are monolithically made on thePlate by embossing (e.g. nanoimprinting) a thin plastic film using amold, and are made of the same materials, where the plastic film areeither PMMA (polymethyl methacrylate) of PS (polystyrene) and thethickness of the Plate is from 50 um to 250 um.

In one preferred embodiment, the spacers are monolithically made on thePlate by embossing (e.g. nanoimprinting) a thin plastic film using amold, and are made of the same materials, where the plastic film areeither PMMA (polymethyl methacrylate) of PS (polystyrene), and thespacers have either a square or rectangle shape, and have the samespacer height.

In one preferred embodiment, the spacers have a square or rectangleshape (with or without round corners).

In one preferred embodiment, the spacers have square or rectanglepillars with the pillar width (spacer width in each lateral direction)between 1 um to 200 um; pillar period (i.e. spacer period) from 2um-2000 um, and pillar height (i.e. spacer height) from 1 um-100 um.

In one preferred embodiment, the spacers made of PMMA or PS have squareor rectangle pillars with the pillar width (spacer width in each lateraldirection) between 1 um to 200 um; pillar period (i.e. spacer period)from 2 um-2000 um, and pillar height (i.e. spacer height) from 1 um-100um.

In one preferred embodiment, the spacers are monolithically made on thePlate and are made of plastic materials, and the spacers have square orrectangle pillars with the pillar width (spacer width in each lateraldirection) between 1 um to 200 um; pillar period (i.e. spacer period)from 2 um-2,000 um, and pillar height (i.e. spacer height) from 1 um-100um.

In one preferred embodiment, the spacers are monolithically made on thePlate and are made of the same materials, and the spacers have square orrectangle pillars with the pillar width (spacer width in each lateraldirection) between 1 um to 200 um; pillar period (i.e. spacer period)from 2 um-2000 um, and pillar height (i.e. spacer height) from 1 um-10um.

In one preferred embodiment, the spacers are monolithically made on thePlate and are made of the same materials selected from PS or PMMA orother plastics, and the spacers have square or rectangle pillars withthe pillar width (spacer width in each lateral direction) between 1 umto 200 um; pillar period (i.e. spacer period) from 2 um-2,000 um, andpillar height (i.e. spacer height) from 10 um-50 um.

Specific sample thickness. In present invention, it was observed that alarger plate holding force (i.e. the force that holds the two platestogether) can be achieved by using a smaller plate spacing (for a givensample area), or a larger sample area (for a given plate-spacing), orboth.

In some embodiments, at least one of the plates is transparent in aregion encompassing the relevant area, each plate has an inner surfaceconfigured to contact the sample in the closed configuration; the innersurfaces of the plates are substantially parallel with each other, inthe closed configuration; the inner surfaces of the plates aresubstantially planar, except the locations that have the spacers; or anycombination of thereof.

Final Sample Thickness and Uniformity. In some embodiments,significantly flat is determined relative to the final sample thickness,and has, depending upon on embodiments and applications, a ratio of tothe sample thickness of less than 0.1%, less than 0.5%, less than 1%,less than 2%, less than 5%, or less than 10%, or a range between any twoof these values.

In some embodiments, flatness relative to the sample thickness can beless than 0.1%, less than 0.5%, less than 1%, less than 2%, less than5%, less than 10%, less than 20%, less than 50%, or less than 100%, or arange between any two of these values.

In some embodiments, significantly flat can mean that the surfaceflatness variation itself (measured from an average thickness) is lessthan 0.1%, less than 0.5%, less than 1%, less than 2%, less than 5%, orless than 10%, or a range between any two of these values. Generally,flatness relative to the plate thickness can be less than 0.1%, lessthan 0.5%, less than 1%, less than 2%, less than 5%, less than 10%, lessthan 20%, less than 50%, or less than 100%, or in a range between anytwo of these values.

The height of the spacers is selected by a desired regulated spacingbetween the plates and/or a regulated final sample thickness and theresidue sample thickness. The spacer height (the predetermined spacerheight), the spacing between the plates, and/or sample thickness is 3 nmor less, 10 nm or less, 50 nm or less, 100 nm or less, 200 nm or less,500 nm or less, 800 nm or less, 1000 nm or less, 1 μm or less, 2 μm orless, 3 μm or less, 5 μm or less, 10 μm or less, 20 μm or less, 30 μm orless, 50 μm or less, 100 μm or less, 150 μm or less, 200 μm or less, 300μm or less, 500 μm or less, 800 μm or less, 1 mm or less, 2 mm or less,4 mm or less, or in a range between any two of the values.

The spacer height, the spacing between the plates, and/or samplethickness is between 1 nm to 100 nm in one preferred embodiment, 100 nmto 500 nm in another preferred embodiment, 500 nm to 1,000 nm in aseparate preferred embodiment, 1 μm (i.e., 1,000 nm) to 2 μm in anotherpreferred embodiment, 2 μm to 3 μm in a separate preferred embodiment, 3μm to 5 μm in another preferred embodiment, 5 μm to 10 μm in a separatepreferred embodiment, and 10 μm to 50 μm in another preferredembodiment, 50 μm to 100 μm in a separate preferred embodiment.

In some embodiments, the spacers can be in spherical beads and randomlydistrusted in a sample.

In some embodiments, the QMAX device is fully transparent or partiallytransparent to reduce the heat absorption by card self, wherein thetransparence is above 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or arange between any two of the values.

In some embodiments, the QMAX device is partially reflective to reducethe heat absorption by card self. wherein the reflectance of the surfaceis above 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or in a range betweenany two of the values.

In some embodiments, the QMAX device and clamp is coated with a heatinsulator layer to reduce the heat absorption by card self. Wherein theheat insulator layer contains materials including the low thermalconductivity material above.

In some embodiments, the clamp covers and seals all the QMAX card inclose configuration.

In some embodiments, the clamp covers and seal only the perimeter of theQMAX card in close configuration.

In some embodiments, the clamp covers and seal only the perimeter of theQMAX card in close configuration, and not the heating and cooling zonearea.

In some embodiments, the clamp covers some of the surface of QMAX cardin close configuration.

In some embodiments, the clamp has a window which is transparent toallow the light go inside the QMAX card and out from the QMAX card.

In some embodiments, the clamp is fully transparent to allow the lightgo inside the QMAX card and out from the QMAX card.

wherein the transparence of the clamp is above 30%, 40%, 50%, 60%, 70%,80%, 90%, 100%, or a range between any two of the values.

In some embodiments, there is air or liquid between the clamp and QMAXdevice in close configuration. In certain embodiments, the liquidincludes but not limit to water, ethane, methane, oil, benzene, Hexane,heptane, silicone oil, polychlorinated biphenyls, liquid air, liquidoxygen, liquid nitrogen etc. In certain embodiments, the gas includesbut not limit to air, argon, helium, nitrogen, oxygen, carbon dioxide,etc.

In some embodiments, after close the clamp, the pressure on QMAX cardsurface applied by the clamp is 0.01 kg/cm², 0.1 kg/cm², 0.5 kg/cm², 1kg/cm², 2 kg/cm², kg/cm², 5 kg/cm², 10 kg/cm², 20 kg/cm², 30 kg/cm², 40kg/cm², 50 kg/cm², 60 kg/cm², 100 kg/cm², 150 kg/cm², 200 kg/cm², 500kg/cm², or a range between any two of the values; and a preferred rangeof 0.1 kg/cm² to 0.5 kg/cm², 0.5 kg/cm² to 1 kg/cm², 1 kg/cm² to 5kg/cm², 5 kg/cm² to kg/cm² (Pressure).

In some embodiments, after close the clamp, the pressure on QMAX cardsurface applied by the clamp is at least 0.01 kg/cm², 0.1 kg/cm², 0.5kg/cm², 1 kg/cm², 2 kg/cm², kg/cm², 5 kg/cm², 10 kg/cm², 20 kg/cm², 30kg/cm², 40 kg/cm², 50 kg/cm², 60 kg/cm², 100 kg/cm², 150 kg/cm², 200kg/cm², or 500 kg/cm²,

As shown in the cross-sectional views of the device in FIG. 23A and FIG.23B, the heating/cooling layer 112 spans across the sample contact area.It should be noted, however, it is also possible that the lateral areaof the heating/cooling layer occupy only a portion of the sample contactarea at a percentage about 1% or more, 5% or more, 10% or more, 20% ormore, 50% or more, 80% or more, 90% or more, 95% or more, 99% or more,85% or less, 75% or less, 55% or less, 40% or less, 25% or less, 8% orless, 2.5% or less. In some embodiments, in order to facilitate thetemperature change of the sample, in some embodiments the lateral areaof the heating/cooling layer is configured so that the sample 90 receivethe thermal radiation from the heating/cooling layer 112 substantiallyuniformly across the lateral dimension of the sample 90 over the samplecontact area.

In some embodiments, the radiation absorbing area is 10%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 100% the total plate area, or a range between anytwo of the values.

In some embodiments, the heating/cooling layer 112 have a thickness of10 nm or more, 20 nm or more, 50 nm or more, 100 nm or more, 200 nm ormore, 500 nm or more, 1 um or more, 2 um or more, 5 um or more, 10 um ormore, 20 um or more, 50 um or more, 100 um or more, 75 um or less, 40 umor less, 15 um or less, 7.5 um or less, 4 um or less, 1.5 um or less,750 nm or less, 400 nm or less, 150 nm or less, 75 nm or less, 40 nm orless, or 15 nm or less, or in a range between any of the two values. Incertain embodiments, the heating/cooling layer 112 have thickness of 100nm or less.

In some embodiments, the area of the sample layer and theheating/cooling layer 112 is substantially larger than the uniformthickness. Here, the term “substantially larger” means that the generaldiameter or diagonal distance of the sample layer and/or theheating/cooling layer is at least 10 time, 15 times, 20 time, 25 times,30 time, 35 times, 40 time, 45 times, 50 time, 55 times, 60 time, 65times, 70 time, 75 times, 80 time, 85 times, 90 time, 95 times, 100time, 150 times, 200 time, 250 times, 300 time, 350 times, 400 time, 450times, 500 time, 550 times, 600 time, 650 times, 700 time, 750 times,800 time, 850 times, 900 time, 950 times, 1u000 time, 1,500 times, 2,000time, 2,500 times, 3,000 time, 3,500 times, 4,000 time, 4,500 times, or5000 time, or in a range between any of the two values.

FIGS. 32A and 32B show exemplary embodiments of the first plate and theheating/cooling layer of the present invention. FIG. 32A is a top viewand FIG. 32B is a sectional view. FIGS. 33A and 33B show sectional viewsof two exemplary embodiments of the present invention, demonstrating thefirst plate, the second plate, and the heating/cooling layer. As awhole, the first plate and the second plate, and optionally theheating/cooling layer, can be viewed as a sample holder, which refers tonot only the embodiments herein shown and/or described, but also otherembodiments that are capable of compressing at least part of a liquidsample into a layer of uniform thickness.

As shown in FIGS. 32A and 32B, in some embodiments, the heating/coolinglayer is in contact with the first plate. It should be noted, however,that in some embodiments the heating/cooling layer can be in contactwith the second plate 20. In addition, in some embodiments theheating/cooling layer is not in contact with any of the plates. In someembodiments, there is no separate structure of the heating/coolinglayer; the first plate and/or the second plate 20 and/or the sampleitself can absorb the electromagnetic radiation some that the sample'stemperature can be raised.

In some embodiments, the heating/cooling layer has an area that is lessthan 1000 mm², 900 mm², 800 mm², 700 mm², 600 mm², 500 mm², 400 mm², 300mm², 200 mm², 100 mm², 90 mm², 80 mm², 75 mm², 70 mm², 60 mm², 50 mm²,40 mm², 30 mm², 25 mm², 20 mm², 10 mm², 5 mm², 2 mm², 1 mm², 0.5 mm²,0.2 mm², 0.1 mm², or 0.01 mm², or in a range between any of the twovalues. In some embodiments, the heating/cooling layer has an area thatis substantially smaller than the area of the first plate (and/or thesecond plate). For example, in certain embodiments, area of theheating/cooling layer occupy only a portion of the area of the firstplate (or the second plate; or the sample contact area of the firstplate or the second plate) at a percentage about 1% or more, 5% or more,10% or more, 20% or more, 50% or more, 80% or more, 90% or more, 95% ormore, 99% or more, 85% or less, 75% or less, 55% or less, 40% or less,25% or less, 8% or less, 2.5% or less.

In some embodiments, the heating/cooling layer has a substantiallyuniform thickness. In some embodiments, the heating/cooling layer has athickness of less than 10 nm, 20 nm, 50 nm, 100 nm, 200 nm, 500 nm, 1um, 2 um, 5 um, 10 um, 20 um, 50 um, 100 um, 200 um, 300 um, 400 um, 500um, 600 um, 700 um, 800 um, 900 um, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm,3.5 mm, 4 mm, 4.5 mm, 5 mm, or 10 mm, or in a range between any of thetwo values.

The heating/cooling layer can take any shape. For example, from a topview the heating/cooling layer can be square, circle, ellipse, triangle,rectangle, parallelogram, trapezoid, pentagon, hexagon, octagon,polygon, or various other shapes.

In some embodiments, the first plate or the second plate has a thicknessof 2 nm or less, 10 nm or less, 100 nm or less, 200 nm or less, 500 nmor less, 1,000 nm or less, 2 μm (micron) or less, 5 μm or less, 10 μm orless, 20 μm or less, 50 μm or less, 100 μm or less, 150 μm or less, 200μm or less, 300 μm or less, 500 μm or less, 800 μm or less, 1 mm(millimeter) or less, 2 mm or less, 3 mm or less, 5 mm or less, 10 mm orless, 20 mm or less, 50 mm or less, 100 mm or less, 500 mm or less, orin a range between any two of these values.

In some embodiments, the first plate and the second plate has a lateralarea of 1 mm² (square millimeter) or less, 10 mm² or less, 25 mm² orless, 50 mm² or less, 75 mm² or less, 1 cm² (square centimeter) or less,2 cm² or less, 3 cm² or less, 4 cm² or less, 5 cm² or less, 10 cm² orless, 100 cm² or less, 500 cm² or less, 1,000 cm² or less, 5,000 cm² orless, 10,000 cm² or less, 10,000 cm² or less, or in a range between anytwo of these values.

In certain embodiments, a fourth power of the inter-spacer-distance(ISD) of the spacers divided by the thickness (h) and the Young'smodulus (E) of the plate (ISD⁴/(hE)) is 5×10⁶ um³/GPa or less;

In certain embodiments, a product of the pillar contact filling factorand the Young's modulus of the spacers is 2 MPa or larger, wherein thepillar contact filling factor is the ratio of the area of the platebeing contacted by the pillars to the entire plate area (in the pillarregion).

In certain embodiments, the spacers have a predetermined substantiallyuniform height and a predetermined constant inter-spacer distance thatis at least about 2 times larger than the size of the analyte, up to 200um, and wherein at least one of the spacers is inside the sample contactarea.

In some embodiments, the plate (either the first plate, the secondplate, or both plates) that has the heating/cooling layer is thin sothat the temperature of the sample can be rapidly changed. For example,in certain embodiments the plate that is in contact with theheating/cooling layer has a thickness equal to or less than 500 um, 200um, 100 um, 50 um, 25 um, 10 um, 5 um, 2.5 um, 1 um, 500 nm, 400 nm, 300nm, 200 nm, or 100 nm, or in a range between any of the two values. Insome embodiments, if only one plate is on contact with theheating/cooling layer, the plate in contact with the heating/coolinglayer is substantially thinner than the plate that is not in contactwith the heating/cooling layer. For example, in some embodiments, thethickness of the plate that is in contact with the heating/cooling layeris less than 1/1,000,000, 1/500,000, 1/100,000, 1/50,000, 1/10,000,1/5,000, 1/1,000, 1/500, 1/100, 1/50, 1/10, 1/5, or ½ of the thicknessof the plate that is in contact with the heating/cooling layer, or in arange between any of the two values.

In some embodiments, the sample layer is thin so that the temperature ofthe sample layer can be rapidly changed. In certain embodiments, thesample layer has a thickness equal to or less than 100 um, 50 um, 25 um,10 um, 5 um, 2.5 um, 1 um, 500 nm, 400 nm, 300 nm, 200 nm, or 100 nm, orin a range between any of the two values.

In various embodiments, the positioning of the heating/cooling layer canalso vary. In some embodiments, as shown in FIG. 33A or 33B, theheating/cooling layer is positioned at the inner surface of the firstplate. Here the inner surface is defined as the surface that is incontact with the sample when the sample is compressed into a layer. Theother surface is the outer surface. In some embodiments, theheating/cooling layer is at the inner surface of the first plate. Insome embodiments, the heating/cooling layer is at the inner surface ofthe second plate. In some embodiments, the heating/cooling layer is atthe outer surface of the first plate. In some embodiments, theheating/cooling layer is inside one or both of the plates. In someembodiments, the heating/cooling layer is at the outer surface thesecond plate. In some embodiments, there are at least twoheating/cooling layers at the inner surfaces and/or outer surfaces ofthe first plate and/or the second plate.

As herein shown and described, in some embodiments, the sample holder isconfigured to compress the fluidic sample into a thin layer, thusreducing the thermal mass of the sample. But reducing the thermal mass,a small amount energy can be able to change the temperature of thesample quickly. In addition, by limiting the sample thickness, thethermal conduction is also limited.

In some embodiments, there is a sample contact area on the respectivesurfaces of the first plate 10 and the second plate 20. The samplecontact area can be any portion of the surface of the first plate 10and/or the second plate 20. In some embodiments, the heating/coolinglayer at least partly overlaps with the sample contact area. In theoverlapping part, the sample is heated quickly due to close proximityand small thermal mass.

In some embodiments, the sample holder 100 is a compressed regulatedopen flow (CROF, also known as QMAX) device, such as but not limited tothe CROF device described in U.S. Provisional Patent Application No.62/202,989, which was filed on Aug. 10, 2015, U.S. Provisional PatentApplication No. 62/218,455, which was filed on Sep. 14, 2015, U.S.Provisional Patent Application No. 62/293,188, which was filed on Feb.9, 2016, U.S. Provisional Patent Application No. 62/305,123, which wasfiled on Mar. 8, 2016, U.S. Provisional Patent Application No.62/369,181, which was filed on Jul. 31, 2016, U.S. Provisional PatentApplication No. 62/394,753, which was filed on Sep. 15, 2016, PCTApplication (designating U.S.) No. PCT/US2016/045437, which was filed onAug. 10, 2016, PCT Application (designating U.S.) No. PCT/US2016/051775,which was filed on Sep. 14, 2016, PCT Application (designating U.S.) No.PCT/US2016/051794, which was filed on Sep. 15, 2016, and PCT Application(designating U.S.) No. PCT/US2016/054025, which was filed on Sep. 27,2016, the complete disclosures of which are hereby incorporated byreference for all purposes.

Edge Sealing for Reducing Sample Evaporation

When the two plates sandwich a sample into a shape with a large lateralto vertical ratio (e.g., 15 mm vs 30 um=500), the evaporation of thesample during a thermal cycling is greatly reduced, since the samplesurfaces covered by the two plate is 500 times larger. Experimentally,we found that in 30 temperature cycling (about 60 secs), there was novisible changes in the sample volume.

On the other hand, in some embodiments, it has a seal element that is incontact with the two plates to form an enclosed chamber which preventssample vapor going out. Such seal element can reduce samplecontamination, in addition to reduce or eliminate sample evaporation.The sealing element can be a tape, plastic seal, oil seal, or acombination of thereof.

In some embodiments, the sealing element does not reach the sample, butthe sealing element is in contact with the two plates to form anenclosed chamber which prevents sample vapor going out. In someembodiments, the sealing element can be used as spacers to regulate therelevant sample's thickness.

In some embodiments, as shown in FIG. 28 , the sample holder 100comprises a sealing element 30 that is configured to seal the spacing102 between the first plate 10 and second plate 20 outside the mediumcontact area at the closed configuration. In certain embodiments, thesealing element 30 encloses the sample 90 within a certain area (e.g.the sample receiving area) so that the overall lateral area of thesample 90 is well defined and measurable. In certain embodiments, thesealing element 30 improves the uniformity of the sample 90, especiallythe thickness of the sample layer.

In some embodiments, as shown in FIG. 28 , the sealing element 30comprises an adhesive applied between the first plate 10 and secondplate 20 at the closed configuration. The adhesive is selective frommaterials such as but not limited to: starch, dextrin, gelatin, asphalt,bitumen, polyisoprene natural rubber, resin, shellac, cellulose and itsderivatives, vinyl derivatives, acrylic derivatives, reactive acrylicbases, polychloroprene, styrene-butadiene, styrene-diene-styrene,polyisobutylene, acrylonitrile-butadiene, polyurethane, polysulfide,silicone, aldehyde condensation resins, epoxide resins, amine baseresins, polyester resins, polyolefin polymers, soluble silicates,phosphate cements, or any other adhesive material, or any combinationthereof. In some embodiments, the adhesive is drying adhesive,pressure-sensitive adhesive, contact adhesive, hot adhesive, or one-partor multi-part reactive adhesive, or any combination thereof. In someembodiments, the glue is natural adhesive or synthetic adhesive, or fromany other origin, or any combination thereof. In some embodiments, theadhesive is spontaneous-cured, heat-cured, UV-cured, or cured by anyother treatment, or any combination thereof.

In some embodiments, as shown in FIG. 28 , the sealing element 30comprises an enclosed spacer (well). For example, the enclosed spacerhas a circular shape (or any other enclosed shape) from a top view andencircle the sample 90, essentially restricting the sample 90 togetherwith the first plate 10 and the second plate 20. In certain embodiments,the enclosed spacer (well) also function as the spacing mechanism 40. Insuch embodiments, the enclosed spacer seals the lateral boundary of thesample 90 as well as regulate the thickness of the sample layer.

In some embodiments, there is an “evaporation-prevention ring” outsideof the liquid area (e.g. sample area) that prevents or reduces the vaporof the liquid escape the card, during a heating.

In some embodiments, there is a clamp outside of the QMAX-card to fixthe QMAX card in its closed configuration during a heating.

In some embodiments, the two plates are compressed by an imprecisepressing force, which is neither set to a precise level norsubstantially uniform. In certain embodiments, the two plates arepressed directly by a human hand.

In some embodiments, the QMAX card/RHC card, including the plates andspacer, is made of the material with low thermal conductivity to reducethe heat absorption by card self.

In some embodiments, there is clamp outside of the QMAX-card to fix theQMAX card in its closed configuration during a heating (namely, theclamp clamps only round the edge of the plates, not the center of theplate pair). In some embodiments, the clamp is made of the material withlow thermal conductivity to reduce the heat absorption by card self.

Heating Source, Extra Heat Sink, Temperature Sensor, and TemperatureControl

The heating layer or the heating/cooling layer in a RHC card isconfigured to be heated by a heating source, wherein the heating sourcedelivers heat energy to the heating/cooling layer optically,electrically, by radio frequency (RF) radiation, or a combinationthereof.

Optical Heating Source. In some embodiments, when a heating layer isheated by a heating source optically, the heating source comprises alight source, that include, but not limited to, LED (light emittingdiode), lasers, lamps, or a combination of thereof.

To get more light from a light source in an optical heating source to aheating layer, some embodiments of the heating sources uses an opticallens, an optical pipe, or a combination thereof.

In some embodiments, the wavelength of the electromagnetic waves is 50nm, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500nm, 550 nm, 600 nm, 650 nm, 700 nm, 750 nm, 800 nm, 850 nm, 900 nm, 950nm, 1 um, 10 um, 25 um, 50 um, 75 um, or 100 um, or in a range betweenany of the two values. In some embodiments, the wavelength of theelectromagnetic waves is 100 nm to 300 nm, 400 nm to 700 nm (visiblerange), 700 nm to 1000 nm (IR range), 1 um to 10 um, 10 um to 100 um, orin a range between any of the two values.

The lens has an NA (numerical aperture) of 0.001, 0.01, 0.05, 0.1, 0.2,0.3, 04, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.5, or in a range betweenany of the two values.

In preferred embodiments, the lens has an NA in a range of 0.01 to 0.1,0.1 to 0.4, 0.4 to 0.7, 0.7 to 1.0, or 1.0 to 1.5.

FIGS. 35A and 35B show a perspective view and a sectional view,respectively, of an embodiment of the present invention, in which anoptical pipe is used to guide the electromagnetic waves (e.g. light)from the heating source (e.g. LED light) to the heating zone or plate.

In certain embodiments, an optical pipe (also termed opticalcollimator), that collimates the light of a light source into theheating zone/plate, comprises a hollow tube with a reflective wall.

One embodiment of an optical pipe comprises a hollow dielectric tubewith a reflective wall (i.e. its inner wall, outer wall, or bothreflective). The hollow dielectric tube can be made of the materials ofglasses, plastics, or a combination. The reflective wall can be a thinlight reflective coating on a wall of the hollow tube. The reflectivecoating can be a thin metal film, such as gold, aluminum, silver,copper, or any mixture or combination thereof. FIG. 38 shows aperspective view of an embodiment of an optical pipe, comprising ahollow tube and a reflective material coating on the outer wall of thetube. A reflective coating also can be in the inside wall. Thereflective wall also can be made of multi-layer interference materialsthat reflect the light of interests. The light pipe can be a materialblock with a hollow pipe that has a reflective wall.

In some embodiments, the hollow pipe has a length in the range of 1 mmto 70 mm, an inner dimension (diameter or width) in the range of 1 mm to40 mm, and a wall thickness in the range of 0.01 mm to 10 mm.

In some preferred embodiments, the hollow pipe for the light pipe has aninner diameter (or an average width) in a range of 1 mm to 5 mm, 5 mm to10 mm, 10 mm to 15 mm, 15 mm to 20 mm, 20 mm to 30 mm, or 30 mm to 50mm.

In some preferred embodiments, the hollow pipe for the light pipe has awall thickness (or an average width) in a range of 0.001 mm to 0.01 mm,0.01 mm to 0.1 mm, 0.1 mm to 0.5 mm, 0.5 mm to 1 mm, 1 mm to 2 mm, or 2mm to 50 mm.

Electrical Heating Source. In some embodiments, when the heating layeror the heating/cooling layer is heated by a heating source electrically,the electric heating source comprises an electrical power supply thatsends an electrical power, though electrical wiring, to theheating/cooling layer.

Extra Heat Sink. In some embodiments, the heat is removed from thesample and the sample holder to the environment, but in someembodiments, extra heat sink will be used to accelerate the heatremoval. The extra heat sink can be a Peltier cooler, passive heatradiator, or both. In some embodiments, fan will be used to create airconvention (directly to the sample and the sample holder, directly toextra heat sink, or both) which accelerate a cooling of the sample.

FIGS. 27A and 27B further show a perspective view and a sectional view,respectively, of some embodiments of the thermal cycling system thatcomprises a sample holder 100 in a closed position and a thermal controlunit 200. Sample holder 100 may include a first plate 10, a second plate20, and a spacing mechanism (not shown). The thermal control unit 200may include a heating source 202 and a controller 204.

As shown in FIG. 27B, the thermal control unit 200 may include a heatingsource 202 and controller 204. In some embodiments, the thermal controlunit 200 provide the energy in the form of electromagnetic waves fortemperature change of the sample.

Referring to both FIGS. 27A and 27B, the heating source 202 isconfigured to project an electromagnetic wave 210 to the heating/coolinglayer 112 of the sample holder 100, which is configured to absorb theelectromagnetic wave 210 and convert a substantial portion of theelectromagnetic wave 210 into heat, resulting in thermal radiation thatelevate the temperature of a portion of the sample 90 that is inproximity of the heating/cooling layer 112. In other words, the couplingof the heating source 202 and the heating/cooling layer 112 isconfigured to generate the thermal energy that is needed to facilitatethe temperature change of the sample 90.

In some embodiments, the radiation from the heating source 202 compriseradio waves, microwaves, infrared waves, visible light, ultravioletwaves, X-rays, gamma rays, or thermal radiation, or any combinationthereof. In some embodiments, the heating/cooling layer 112 has apreferred range of light wavelength at which the heating/cooling layer112 has a high absorption efficiency. In some embodiments, the heatingsource 202 is configured to project the electromagnetic wave at awavelength range within, overlapping with, or enclosing the preferredwavelength range of the heating/cooling layer 112. In other embodiments,in order to facilitate the temperature change, the wavelength isrationally designed away from the preferred wavelength of theheating/cooling layer.

In some embodiments, the heating source 202 comprise a laser sourceproviding a laser light within a narrow wavelength range. In otherembodiments, the heating source 202 comprises a LED (light-emittingdiode) of a plurality thereof.

Temperature sensors. The temperature of the sample can be controlled bydelivering pre-calibrated energy to the heating zone/layer with a realtime temperature sensor, by using a real time temperature sensor, orboth.

A real time temperature sensor can be thermometer, thermal couple,radiation temperature sensor, temperature sensitive dye (which changeeither light intensity or color or both with temperature), or acombination thereof.

As shown in FIG. 28 , in some embodiments the thermal control unit 200comprises a thermometer 206. In some embodiments, the thermometer 206provides a monitoring and/or feedback mechanism tocontrol/monitor/adjust the temperature of the sample 90. For example, insome embodiments the thermometer 206 is configured to measure thetemperature at or in proximity of the sample contact area. In certainembodiments, the thermometer 206 is configured to directly measure thetemperature of the sample 90. In some embodiments, the thermometer 206is selected from the group consisting of: fiber optical thermometer,infrared thermometer, fluidic crystal thermometer, pyrometer, quartzthermometer, silicon bandgap temperature sensor, temperature strip,thermistor, and thermocouple. In certain embodiments, the thermometer206 is an infrared thermometer.

In some embodiments, the thermometer 206 is configured to send signalsto the controller 204. Such signals comprise information related to thetemperature of the sample 90 so that the controller 204 makescorresponding changes. For example, during a PCR, for the denaturationstep the target temperature is set for 95° C.; after measurement, thethermometer sends a signal to the controller 204, indicating that themeasured temperature of the sample 90 is actually 94.8° C.; thecontroller 204 thus alters the output the heating source 202, whichprojects an electromagnetic wave or adjust particular parameters (e.g.,intensity or frequency) of an existing electromagnetic wave so that thetemperature of the sample 90 is increased to 95° C. Suchmeasurement-signaling-adjustment loop is applied to any step in anyreaction/assay.

Controllers. Referring to panels (A) and (B) of FIG. 25 , the controller204 is configured to control the electromagnetic wave 210 projected fromthe heating source 202 for the temperature change of the sample. Theparameters of the electromagnetic wave 210 that the controller 204controls include, but are not limited to, the presence, intensity,wavelength, incident angle, and any combination thereof. In someembodiments, the controller is operated manually, for instance, it is assimple as a manual switch that controls the on and off of the heatingsource, and therefore the presence of the electromagnetic wave projectedfrom the heating source. In other embodiments, the controller includeshardware and software that are configured to control the electromagneticwave automatically according to one or a plurality of pre-determinedprograms.

In some embodiments, the pre-determined program refers to a schedule inwhich the parameter(s) (e.g., presence, intensity, and/or wavelength) ofthe electromagnetic wave 210 is/are set to pre-determined levels forrespective pre-determined periods of time. In other embodiments, thepre-determined program refers to a schedule in which the temperature ofthe sample 90 is set to pre-determined levels for respectivepre-determined periods of time and the time periods for the change ofthe sample temperature from one pre-determined level to anotherpre-determined level are also set respectively. In some embodiments, thecontroller 204 is configured to be programmable, which means thecontroller 204 comprises hardware and software that are configured toreceive and carry out pre-determined programs for the system that aredelivered by the operator of the system.

FIG. 28 shows a sectional view of an embodiment of the presentinvention, demonstrating the thermal cycler system and showingadditional elements that facilitates temperature change and control. Asshown in FIG. 28 , the thermal cycler system comprises a sample holder100 and a thermal control unit 200. The sample holder 100 comprises afirst plate 10, a second plate 20, a spacing mechanism 40, and a sealingelement 30; the thermal control unit 200 comprises a heating source 202,a controller 204, a thermometer 206, and an expander 208.

FIG. 28 shows the sample holder 100 in a closed configuration, in whichthe inner surfaces 11 and 21 of the first and second plates 10 and 20face each other and the spacing 102 between the two plates are regulatedby a spacing mechanism 40. If a sample 90 has been deposited on one orboth of the plates in the open configuration, when switching to theclosed configuration, the first plate 10 and the second plate 20 arepressed by a human hand or other mechanisms, the sample 90 is thuscompressed by the two plates into a thin layer. In some embodiments, thethickness of the layer is uniform and the same as the spacing 102between the two plates. In certain embodiments, the spacing 102 (andthus the thickness of the sample layer) is regulated by the spacingmechanism 40. In some embodiments, the spacing mechanism comprises anenclosed spacer that is fixed to one of the plates. In some embodiments,the spacing mechanism 40 comprises a plurality of pillar shaped spacersthat are fixed to one or both of the plates. Here the term “fixed” meansthat the spacer(s) is attached to a plate and the attachment ismaintained during at least a use of the plate.

In some embodiments, the controller 204 is configured to adjust thetemperature of the sample to facilitate an assay and/or reactioninvolving the sample 90 according to a pre-determined program. In someembodiments, the assay and/or reaction is a PCR. In certain embodiments,the controller 204 is configured to control the presence, intensity,and/or frequency of the electromagnetic wave from the heating source206.

Sample Signal Monitoring

As shown in FIGS. 32 and 33 , a signal sensor can be used to detect thesignal from the sample (and the products from a reaction during atemperature change) in the sample holder.

In some embodiments, the signal sensor is an optical sensor that isconfigured to image the fluidic sample. For example, optical sensor is aphotodetector, camera, or a device capable of capturing images of thefluidic sample. In some embodiments, the optical sensor can be a camera.In some embodiments, the camera is a camera integrated into a mobiledevice (e.g. a smartphone or tablet computer). In some embodiments, thecamera is separated from other parts of the system. In some embodiments,a light source or multi light sources are used to excite the sample (andthe products from a reaction during a temperature change) for generatinga signal

In some embodiments, the signal sensor is an electrical sensor that isconfigured to detect electrical signals from the device. In someembodiments, the signal sensor is a mechanical sensor that is configuredto detect mechanical signals from the device.

In some embodiments, the signal sensor is configured to monitor theamount of an analyte in the sample. In some embodiments, the signalsensor is outside the chamber and receive optical signals from thesample through an optical aperture on the chamber.

Base and Systems

In some embodiments, the apparatus further comprises a base (an adaptor)that is configured to house the sample card, the heating source,temperature sensors, a part of an entire of temperature controlled(include a smartphone in some embodiments), extra-heat sink(optionally), a fan (optionally) or a combination of thereof. In someembodiments, the adaptor comprises a card slot, into which the samplecard can be inserted. In some embodiments, the sample card, after beingfully inserted into the slot, or after reaching a pre-defined positionin the slot, is stabilized and stays in place without any movement.

In some embodiments, the base (adaptor) is configured to position thesample card, and the sample within the sample card, in the field of viewof an optical sensor (e.g. a camera) so that the sample can be imaged.In certain embodiments, the camera is part of a mobile device (e.g. asmartphone). In some embodiments, the adaptor comprises a slider in theslot. In certain embodiments, the sample card can be put onto theslider, which can slide into or out of the slot in the adaptor. In someembodiments, the adaptor comprises a card support. In certainembodiments, the sample card can be put on the card support, which doesnot need to be moved before imaging.

In some embodiments, the adaptor is configured to be connectable to anoptical sensor so that the relative position of the optical sensor(e.g., mobile device; e.g., smartphone) and the sample card is fixed. Incertain embodiments, the adaptor can include a connecting member that isreplaceable and directly attach to the mobile device (as an example).The connecting member can be slid onto the mobile device and firmlyattach the adaptor to the mobile device, optimally positioning thesample card to be imaged or for the detection and/or measurement of theanalyte. In certain embodiments, the connecting member is replaceable sothat different connecting members can be used for different mobiledevices.

In some embodiments, the adaptor comprises a radiation aperture thatallows the passage of the electromagnetic waves that heat or cool thesample. In some embodiments, the adaptor comprises an optical aperturethat allows imaging of the sample. In some embodiments, the adaptorserves as a heating sink for the sample card. FIGS. 34 and 35 provideadditional embodiments of the system. FIG. 34 shows a sectional view ofan exemplary embodiment of the present invention, demonstrating thesystem to rapidly change the temperature of a sample. FIG. 34 shows thedetailed elements of a heating source according to one embodiment.

As shown in FIG. 34 and FIG. 35 , in some embodiments, the systemcomprises a sample holder and a heating source. In some embodiments, thesample holder comprises the first plate, the second plate, and/or theheating/cooling layer, as herein described. The heating source emitselectromagnetic waves that reach the sample and can be converted to heatthat raises the temperature of the sample. In some embodiments, theconversion is conducted by the heating/cooling layer. When there is nospecific heating/cooling layer, the conversion is conducted by otherparts of the sample holder.

As shown in FIG. 34 and FIG. 35 , in some embodiments, the systemcomprises a chamber that encages the sample holder. In some embodiments,the chamber is an example of the extra heat sink in FIG. 22 . In someembodiments, the chamber comprises an optical aperture that isconfigured to allow imaging of the sample. In some embodiments, thechamber comprises a radiation aperture configured to allow passage ofelectromagnetic waves from a heating source to the heating/coolinglayer. In certain embodiments, a window is positioned at the radiationaperture to allow the passage of the electromagnetic waves. In certainembodiments, a filter (e.g. bandpass filter) is positioned at theoptical aperture to allow the imaging of the sample in the sampleholder.

In some embodiments, the chamber is used to absorb the heat from thesample and/or the heating source. In some embodiments, the chambercomprises a metal case. In some embodiments, the chamber comprises anouter layer. In certain embodiments, the outer layer is black. In someembodiments, the outer layer is made from black metal. In someembodiments, the chamber comprises an inner layer. In some embodiments,the inner layer is made from non-reflective material. In certainembodiments, the inner layer is black. In some embodiments, the innerlayer is made from black metal.

As shown in FIG. 34 and FIG. 35 , in some embodiments, the systemcomprises an optical sensor, which is configured to capture images ofthe fluidic sample in the sample holder. In some embodiments, the systemfurther comprises a light source, which in some cases can be integratedwith the optical sensor and in some cases can be separate. In someembodiments, the light source is configured to provide excitation lightthat can reach the sample. In some embodiments, the sample can providesignal light that can be captured by the optical sensor so that imagesare taken.

As shown in FIG. 34 , in some embodiments, the heating source comprisesan LED or laser diode. In certain embodiments, the heating sourcefurther comprises a fiber coupler and a fiber that direct the light fromthe LED/Laser diode to the sample holder.

FIG. 35 shows a sectional view of an exemplary embodiment of the presentinvention, demonstrating the system to rapidly change the temperature ofa sample. FIG. 35 shows the detailed elements of a heating sourceaccording to one embodiment. As shown in FIG. 35 , in some embodiments,the heating source comprises an LED or laser diode. In certainembodiments, the heating source further comprises one or more focusinglenses that focuses the electromagnetic waves from the heating source tothe sample in the sample holder.

As shown in FIG. 28 , the thermal control unit 200 comprises a beamexpander 208, which is configured to expand the electromagnetic wavefrom the heating source 202 from a smaller diameter to a largerdiameter. In some embodiments, the electromagnetic wave projected fromthe heating source 202 is sufficient to cover the entire sample contactarea; in some embodiments however, it is necessary to expand the coveredarea of the electromagnetic wave projected directed from the heatingsource 202 to produce an expanded electromagnetic wave 210, providing aheat source for all the sample contact area(s). The beam expander 208employs any known technology, including but not limited to the beamexpanders described in U.S. Pat. Nos. 4,545,677, 4,214,813, 4,127,828,and 4,016,504, and U.S. Pat. Pub. No. 2008/0297912 and 2010/0214659,which are incorporated by reference in their entireties for allpurposes.

Smartphone

In some embodiments, the sample card is imaged by a mobile device. Incertain embodiments, the mobile device is a smartphone, which can serveas an example.

In some embodiments, the smartphone comprises a camera that can be usedto image the sample in the sample card. In some embodiments, an adaptoris used to accommodate the sample card and the adaptor is configured toattach to the smartphone so that the sample card (and the sampletherein) can be placed in the field of view of the camera.

In some embodiments, the smartphone can also serve as the control unit,which is configured to control the apparatus. For example, thesmartphone can be used control the heating and/or cooling of the samplecard. In certain embodiments, the smartphone is connected to the heatingsource and controls the electromagnetic waves from the heating source.In some embodiments, the smartphone controls the presence, intensity,wavelength, frequency, and/or angle of the electromagnetic waves. Incertain embodiments, the smartphone receives the temperature data from athermometer that measures the temperature of the sample. In certainembodiments, the smartphone controls the electromagnetic waves based onthe temperature data.

In some embodiments, the smartphone can also serve as a data processingand communication device. For example, after the sample has been imaged,the images can be saved in the smart phone. In certain embodiments, thesave images can be processed by software or applications in thesmartphone. For example, the presence and/or amount of the analyte canbe deduced from the images by software or applications in thesmartphone. In certain embodiments, the processed results can bedisplayed on the screen of the smart phone. In certain embodiments, theprocessed results can be sent to the user, e.g. with email or othermessaging software. In certain embodiments, the processed results can besent to a third party, e.g., a healthcare professional, who can makefurther diagnostics and/or process the data in additional steps. In someembodiments, the images, without processing, can be displayed and/ortransmitted. In certain embodiments, the images are displayed on thescreen of the smartphone. In certain embodiments, the images are sent tothe user, e.g. by email or other messaging software. In certainembodiments, the images can be sent to a third party, e.g. a remoteserver, which can process the images further. In some embodiments, theresults and/or images are compressed and/or encrypted before being sent.

Use of RHC Card

The RHC card in the description can be used as one step of multiplesteps in test a sample, or as one step that perform entire test.

In some embodiments, a RHC card is used in a so-called “one-step assay”,wherein all reagents and a sample for an analysis are loaded on a RHCcard and a thermal cycling or temperature change is performed and thesignal is being observed during the thermal cycling or temperaturechange.

OTHER EMBODIMENTS Embodiment 1

One embodiment comprises a device of the embodiment SH-1 to SH-6,wherein the first plate and the second plate are flexible plastic filmand/or thin glass film, that each has a substantially uniform thicknessof a value selected from a range between 1 um to 25 um.

Each plate has an area in a range of 1 cm² to 16 cm².

The sample sandwiched between the two plate has a thickness of 40 um orless.

The relevant sample to the entire sample ratio (RE ratio) is 12% orless.

The cooling zone is at least 9 times larger than the heating zone.

The sample to non-sample thermal mass ratio is 2.2 or lager.

The RHC have no spacer in some embodiments, but do have spacers in otherembodiments.

STC ratio is and the cooling zone comprises a layer of the material thathas a thermal conductivity of 70 W/m-K or higher and a thermalconductivity times its thickness.

Embodiment 2

For the embodiments of SH-1 to SH-x, they have the following parameterarrange for fast thermal cycling.

The first plate and second plates are plastic or a thin glass. The firstplate and second plate have a thickness of 100 nm, 500 nm, 1 um, 5 um,10 um, in a range between any of the two values.

The sample between the two plates has a thickness of 5 um, 10 um, 30 um,50 um, 100 um, or in a range between any of the two values.

The distance from the H/C layer to the sample is 10 nm, 100 nm, 500 nm,1 um, 5 um, 10 um, or in a range between any of the two values.

The ratio of the cooling zone area to the relevant sample area is 16, 9,4, 2, or in a range between any of the two values.

The ratio of the cooling zone area to the heating area is 16, 9, 4, 2,or in a range between any of the two values.

The distance between the H/C layer and the heating source (e.g. LED) is5 mm, 10 mm, 20 mm, 30 mm, or in a range between any of the two values.

Embodiment 3

For the embodiments of SH-1 to SH-x, they have the following parameterarrange for fast thermal cycling.

The first plate and second plates are plastic or a thin glass. The firstplate has a thickness of 10 um, 25 um, 50 um, or in a range between anyof the two values; while the second plate (that plate that has heatinglayer or cooling layer) has a thickness of 100 nm, 500 nm, 1 um, 5 um,10 um, in a range between any of the two values.

The sample between the two plates has a thickness of 5 um, 10 um, 30 um,50 um, 100 um, or in a range between any of the two values.

The distance between the H/C layer and the sample is 10 nm, 100 nm, 500nm, 1 um, 5 um, 10 um, or in a range between any of the two values.

The ratio of the cooling zone area to the relevant sample area is 16, 9,4, 2, or in a range between any of the two values.

The ratio of the cooling zone area to the heating area is 16, 9, 4, 2,or in a range between any of the two values.

The distance between the H/C layer and the heating source (e.g. LED) is5 mm, 10 mm, 20 mm, 30 mm, or in a range between any of the two values.

Embodiment 4

For the embodiments of SH-1 to SH-x, they have the following parameterarrange for fast thermal cycling.

The first plate and second plates are plastic or a thin glass. The firstplate and second plate have a thickness of 100 nm, 500 nm, 1 um, 5 um,10 um, 25 um, 50 um, 100 um, 175 um, 250 um, or in a range between anyof the two values.

The sample between the two plates has a thickness of 100 nm, 500 nm, 1um, 5 um, 10 um, 25 um, 50 um, 100 um, 250 um, or in a range between anyof the two values.

The distance between the H/C layer and the sample is 100 nm, 500 nm, 1um, 5 um, 10 um, 25 um, 50 um, 100 um, 175 um, 250 um, or in a rangebetween any of the two values.

The ratio of the cooling zone area to the relevant sample area is 100,64, 16, 9, 4, 2, 1, 0.5, 0.1, or in a range between any of the twovalues.

The ratio of the cooling zone area to the heating zone is 100, 64, 16,9, 4, 2, 1, 0.5, 0.1, or in a range between any of the two values.

The distance between the H/C layer and the heating source (e.g. LED) is500 um, 1 mm, 3 mm, 5 mm, 10 mm, 20 mm, 30 mm, or in a range between anyof the two values.

Embodiment 5

For the embodiments of SH-1 to SH-5, they have the following parameterarrange for fast thermal cycling.

A light pipe collimates the light from a light source (e.g. LED) intothe heating zone. The light pile comprises a structure with a hollowhole (e.g. a tube or a structure milled a hole) with a reflective wall.The light pile has a lateral dimension for 1 mm to 8 mm and length of 2mm to 5o mm.

Embodiment 6

For the embodiments of SH-1 to SH-5, they have the following parameterarrange for fast thermal cycling.

The first plate and second plates are plastic or a thin glass. The firstplate and second plate have a thickness of 100 nm, 500 nm, 1 um, 5 um,10 um, in a range between any of the two values.

The sample between the two plates has a thickness in a range of 1 to 5um, 5 um to 10 um, 10 to 30 um, or 30 um to 50 um.

The distance from the H/C layer to the sample is in a range of 10 nm to100 nm, 100 nm to 500 nm, 500 nm to 1 um, 1 um to 5 um, 5 um to 10 um,or 10 um to 25 um.

The ratio of the cooling zone area to the relevant sample area is 16, 9,4, 2, or in a range between any of the two values.

The ratio of the cooling zone area to the heating area is 16, 9, 4, 2,or in a range between any of the two values.

The distance between the H/C layer and the heating source (e.g. LED) is5 mm, 10 mm, 20 mm, 30 mm, or in a range between any of the two values.

The KC ratio for the cooling layer is in a range of between 0.5 cm²/secand 0.7 cm²/sec, 0.7 cm²/sec and 0.9 cm²/sec, 0.9 cm²/sec and 1 cm²/sec,1 cm²/sec and 1.1 cm²/sec, 1.1 cm²/sec and 1.3 cm²/sec, 1.3 cm²/sec and1.6 cm²/sec, 1.6 cm²/sec and 2 cm²/sec, or 2 cm²/sec and cm²/sec.

The sample to non-sample thermal mass ratio is in a range of between 0.2to 0.5, 0.5 to 0.7, 0.7 to 1, 1 to 1.5, 1.5 to 5, 5 to 10, 10 to 30, 30to 50, or 50 to 100.

Embodiment 7

For the embodiments of SH-1 to SH-5, as well as Embodiments 1 toEmbodiments 6, they have the following parameter arrange for fastthermal cycling:

The first plate and/or the second plate has a lateral area in a range of1 mm² (square millimeter) to 10 mm², 10 mm² to 50 mm², 50 mm² to 100mm², 1 cm² to 5 cm², 5 cm² to 20 cm², or 20 cm² to 50 cm².

The scaled thermal conduction ratio (STM ratio) is in a range of between10 to 20, 30 to 50, 50 to 70, 70 to 100, 100 to 1,000, 1,000 to 10,000,or 10,000 to 1,000,000; and the cooling zone (layer) has thermalconductivity times its thickness of 6×10⁻⁵ W/K, 9×10⁻⁵ W/K, 1.2×10⁻⁴W/K, 1.5×10⁻⁴ W/K, 1.8×10⁻⁴ W/K, 2.1×10⁻⁴ W/K, 2.7×10⁻⁴ W/K, 3×10⁻⁴ W/K,1.5×10⁻⁴ W/K, or in a range between any of the two values.

The sample holder (RHC card) has not significant thermal conduction tothe environment during a thermal cycling.

Sample Types

The devices, systems, and methods herein disclosed can be used forsamples such as but not limited to diagnostic sample, clinical sample,environmental sample and foodstuff sample. The types of sample includebut are not limited to the samples listed, described and summarized inPCT Application (designating U.S.) Nos. PCT/US2016/045437 andPCT/US0216/051775, which were respectively filed on Aug. 10, 2016 andSep. 14, 2016, and are hereby incorporated by reference by theirentireties.

For example, in some embodiments, the devices, systems, and methodsherein disclosed are used for a sample that includes cells, tissues,bodily fluids and/or a mixture thereof. In some embodiments, the samplecomprises a human body fluid. In some embodiments, the sample comprisesat least one of cells, tissues, bodily fluids, stool, amniotic fluid,aqueous humour, vitreous humour, blood, whole blood, fractionated blood,plasma, serum, breast milk, cerebrospinal fluid, cerumen, chyle, chime,endolymph, perilymph, feces, gastric acid, gastric juice, lymph, mucus,nasal drainage, phlegm, pericardial fluid, peritoneal fluid, pleuralfluid, pus, rheum, saliva, sebum, semen, sputum, sweat, synovial fluid,tears, vomit, urine, and exhaled breath condensate.

In some embodiments, the devices, systems, and methods herein disclosedare used for an environmental sample that is obtained from any suitablesource, such as but not limited to: river, lake, pond, ocean, glaciers,icebergs, rain, snow, sewage, reservoirs, tap water, drinking water,etc.; solid samples from soil, compost, sand, rocks, concrete, wood,brick, sewage, etc.; and gaseous samples from the air, underwater heatvents, industrial exhaust, vehicular exhaust, etc. In certainembodiments, the environmental sample is fresh from the source; incertain embodiments, the environmental sample is processed. For example,samples that are not in liquid form are converted to liquid form beforethe subject devices, systems, and methods are applied.

In some embodiments, the devices, systems, and methods herein disclosedare used for a foodstuff sample, which is suitable or has the potentialto become suitable for animal consumption, e.g., human consumption. Insome embodiments, a foodstuff sample includes raw ingredients, cooked orprocessed food, plant and animal sources of food, preprocessed food aswell as partially or fully processed food, etc. In certain embodiments,samples that are not in liquid form are converted to liquid form beforethe subject devices, systems, and methods are applied.

The subject devices, systems, and methods can be used to analyze anyvolume of the sample. Examples of the volumes include, but are notlimited to, about 10 mL or less, 5 mL or less, 3 mL or less, 1microliter (μL, also “uL” herein) or less, 500 μL or less, 300 μL orless, 250 μL or less, 200 μL or less, 170 μL or less, 150 μL or less,125 μL or less, 100 μL or less, 75 μL or less, 50 μL or less, 25 μL orless, 20 μL or less, 15 μL or less, 10 μL or less, 5 μL or less, 3 μL orless, 1 μL or less, 0.5 μL or less, 0.1 μL or less, 0.05 μL or less,0.001 μL or less, 0.0005 μL or less, 0.0001 μL or less, 10 μL or less, 1μL or less, or a range between any two of the values.

In some embodiments, the volume of the sample includes, but is notlimited to, about 100 μL or less, 75 μL or less, 50 μL or less, 25 μL orless, 20 μL or less, 15 μL or less, 10 μL or less, 5 μL or less, 3 μL orless, 1 μL or less, 0.5 μL or less, 0.1 μL or less, 0.05 μL or less,0.001 μL or less, 0.0005 μL or less, 0.0001 μL or less, 10 pL or less, 1pL or less, or a range between any two of the values. In someembodiments, the volume of the sample includes, but is not limited to,about 10 μL or less, 5 μL or less, 3 μL or less, 1 μL or less, 0.5 μL orless, 0.1 μL or less, 0.05 μL or less, 0.001 μL or less, 0.0005 μL orless, 0.0001 μL or less, 10 pL or less, 1 pL or less, or a range betweenany two of the values.

In some embodiments, the amount of the sample is about a drop of liquid.In certain embodiments, the amount of sample is the amount collectedfrom a pricked finger or fingerstick. In certain embodiments, the amountof sample is the amount collected from a microneedle, micropipette or avenous draw.

In certain embodiments, the sample holder is configured to hold afluidic sample. In certain embodiments, the sample holder is configuredto compress at least part of the fluidic sample into a thin layer. Incertain embodiments, the sample holder comprises structures that areconfigured to heat and/or cool the sample. In certain embodiments, theheating source provides electromagnetic waves that can be absorbed bycertain structures in the sample holder to change the temperature of thesample. In certain embodiments, the signal sensor is configured todetect and/or measure a signal from the sample. In certain embodiments,the signal sensor is configured to detect and/or measure an analyte inthe sample. In certain embodiments, the heat sink is configured toabsorb heat from the sample holder and/or the heating source. In certainembodiments, the heat sink comprises a chamber that at least partlyenclose the sample holder.

Applications

The devices, systems, and methods herein disclosed can be used invarious types of biological/chemical sampling, sensing, assays andapplications, which include the applications listed, described andsummarized in PCT Application (designating U.S.) No. PCT/US2016/045437,which was filed on Aug. 10, 2016, and is hereby incorporated byreference by its entirety.

In some embodiments, the devices, systems, and methods herein disclosedare used in a variety of different application in various field, whereindetermination of the presence or absence, quantification, and/oramplification of one or more analytes in a sample are desired. Forexample, in certain embodiments the subject devices, systems, andmethods are used in the detection of proteins, peptides, nucleic acids,synthetic compounds, inorganic compounds, and other molecules,compounds, mixtures and substances. The various fields in which thesubject devices, systems, and methods can be used include, but are notlimited to: diagnostics, management, and/or prevention of human diseasesand conditions, diagnostics, management, and/or prevention of veterinarydiseases and conditions, diagnostics, management, and/or prevention ofplant diseases and conditions, agricultural uses, food testing,environments testing and decontamination, drug testing and prevention,and others.

The applications of the present invention include, but are not limitedto: (a) the detection, purification, quantification, and/oramplification of chemical compounds or biomolecules that correlates withcertain diseases, or certain stages of the diseases, e.g., infectiousand parasitic disease, injuries, cardiovascular disease, cancer, mentaldisorders, neuropsychiatric disorders and organic diseases, e.g.,pulmonary diseases, renal diseases, (b) the detection, purification,quantification, and/or amplification of cells and/or microorganism,e.g., virus, fungus and bacteria from the environment, e.g., water,soil, or biological samples, e.g., tissues, bodily fluids, (c) thedetection, quantification of chemical compounds or biological samplesthat pose hazard to food safety, human health, or national security,e.g. toxic waste, anthrax, (d) the detection and quantification of vitalparameters in medical or physiological monitor, e.g., glucose, bloodoxygen level, total blood count, (e) the detection and quantification ofspecific DNA or RNA from biological samples, e.g., cells, viruses,bodily fluids, (f) the sequencing and comparing of genetic sequences inDNA in the chromosomes and mitochondria for genome analysis or (g) thedetection and quantification of reaction products, e.g., duringsynthesis or purification of pharmaceuticals.

In some embodiments, the subject devices, systems, and methods are usedin the detection of nucleic acids, proteins, or other molecules orcompounds in a sample. In certain embodiments, the devices, systems, andmethods are used in the rapid, clinical detection and/or quantificationof one or more, two or more, or three or more disease biomarkers in abiological sample, e.g., as being employed in the diagnosis, prevention,and/or management of a disease condition in a subject. In certainembodiments, the devices, systems, and methods are used in the detectionand/or quantification of one or more, two or more, or three or moreenvironmental markers in an environmental sample, e.g. sample obtainedfrom a river, ocean, lake, rain, snow, sewage, sewage processing runoff,agricultural runoff, industrial runoff, tap water or drinking water. Incertain embodiments, the devices, systems, and methods are used in thedetection and/or quantification of one or more, two or more, or three ormore foodstuff marks from a food sample obtained from tap water,drinking water, prepared food, processed food or raw food.

In some embodiments, the devices, systems and methods of the inventioncan be used to detect an analyte. In some embodiments, the analyte is apathogen. Exemplary pathogens that can be detected include, but are notlimited to: Varicella zoster; Staphylococcus epidermidis, Escherichiacoli, methicillin-resistant Staphylococcus aureus (MSRA), Staphylococcusaureus, Staphylococcus hominis, Enterococcus faecalis, Pseudomonasaeruginosa, Staphylococcus capitis, Staphylococcus warneri, Klebsiellapneumoniae, Haemophilus influenzae, Staphylococcus simulans,Streptococcus pneumoniae and Candida albicans; gonorrhea (Neisseriagorrhoeae), syphilis (Treponena pallidum), chlamydia (Chlamydiatracomitis), nongonococcal urethritis (Ureaplasm urealyticum), chancroid(Haemophilus ducreyi), trichomoniasis (Trichomonas vaginalis);Pseudomonas aeruginosa, methicillin-resistant Staphlococccus aureus(MSRA), Klebsiella pneumoniae, Haemophilis influenzae, Staphylococcusaureus, Stenotrophomonas maltophilia, Haemophilis parainfluenzae,Escherichia coli, Enterococcus faecalis, Serratia marcescens,Haemophilis parahaemolyticus, Enterococcus cloacae, Candida albicans,Moraxiella catarrhalis, Streptococcus pneumoniae, Citrobacter freundii,Enterococcus faecium, Klebsella oxytoca, Pseudomonas fluorscens,Neiseria meningitidis, Streptococcus pyogenes, Pneumocystis carinii,Klebsella pneumoniae, Legionella pneumophila, Mycoplasma pneumoniae, andMycobacterium tuberculosis, etc.

In some embodiments, the devices, systems and methods of the inventioncan be used to detect an analyte that is a diagnostic marker. In someembodiments, the diagnostic marker is selected from any of the followingTables.

TABLE 4.1 Diagnostic markers Marker(s) Disease(s), Disorder(s), (sourceof sample) or Condition(s) Aβ42, amyloid Alzheimer's disease (AD)beta-protein (cerebrospinal fluid, CSF) Fetuin-A (CSF) Multiplesclerosis (MS) Tau (CSF) Niemann-pick type C, parkinsonian disorders(neurodegenerative disorders) Secretogranin II (CSF) Bipolar disorderPrion protein (CSF) AD, prion disease Cytokines (CSF) HIV-associatedneurocognitive disorders Alpha-synuclein (CSF) Parkinsonian disorders(neurodegenerative disorders) Neurofilament Axonal degeneration lightchain (CSF) Parkin (CSF) Neurodegenerative disorders PTEN inducedNeurodegenerative disorders putative kinase 1 (CSF) DJ-1 (CSF)Neurodegenerative disorders Leucine-rich Neurodegenerative disordersrepeat kinase (CSF) Mutated ATP12A2 Kufor-Rakeb disease (CSF) Apo H(CSF) Parkinson's disease (PD) Ceruloplasmin (CSF) PD Peroxisomeproliferator- PD activated receptor gamma coactivator- 1-alpha (PGC-1α)(CSF) Transthyretin (CSF) CSF rhinorrhea (nasal surgery samples)Vitamin-D binding MS progression protein (CSF) Proapoptotic kinase AD R(PKR) and its phosphorylated PKR (pPKR)(CSF) CXCL13 (CSF) MS IL-12p40,CXCL13 Intrathecal inflammation and IL-8 (CSF) Dkk-3 (semen) Prostatecancer p14 endocan Sepsis, lung inflammatory reaction fragment (blood)Serum (blood) Neuromyelitis optica ACE2 (blood) Cardiovascular diseaseAutoantibody to Early diagnosis of esophageal CD25 (blood) squamous cellcarcinoma hTERT (blood) Lung cancer CAI25 (MUC 16) Lung cancer (blood)VEGF (blood) Lung cancer sIL-2 (blood) Lung cancer Osteopontin (blood)Lung cancer Human epididymis Ovarian cancer protein 4 (HE4)(blood)Alpha-fetal Pregnancy protein (blood) Albumin (urine) Diabetes Albumin(urine) uria Albuminuria Microalbuminuria Kidney leaks (urine) AFP(urine) Mirror fetal AFP levels Neutrophil Acute kidney injurygelatinase-associated lipocalin (NGAL) (urine) Interleukin-18 Acutekidney injury (IL-18)(urine) Kidney injury Acute kidney injurymolecule-1 (KIM-1)(urine) Liver fatty acid Acute kidney injury bindingprotein (L-FABP)(urine) LMP1 (saliva) Epstein-Barr virus oncoprotein(nasopharyngeal carcinomas) BARF1 (saliva) Epstein-Barr virusoncoprotein (nasopharyngeal carcinomas) Interleukin-8 Oral cancer,spinalcellular (IL-8)(saliva) carcinoma Carcinoembryonic Oral orsalivary malignant tumors antigen (CEA)(saliva) BRAF, CCNI, Lung cancerEGRF, FGF19, FRS2, GREB1, and LZTS1 (saliva) CA 125 (saliva) Ovariancancer Thioredoxin (saliva) Spinalcellular carcinoma Beta-2microglobulin HIV (saliva) Tumor necrosis HIV factor-alpha receptors(saliva) CA15-3 (saliva) Breast cancer

TABLE 4.2 Diagnostic markers HPA axis activity (Cushing’s disease,Adrenal cortex diseases, etc.): Cortisol Pregnancy/fetal development:Progesterone, human chorionic gonadotropin, Levonorgestrel,alpha-fetoprotein, early conception factor, Unconjugated Estriol,Estradiol, interleukin-6, Inhibin-A Infant development: NGAL, KIM-1,Cys-C, and B2mG, AFP, S100B, MBP Menopause: Follicle stimulating hormone(FSH), Estrogen and progesterone, testosterone, free testosterone, anddehydroepiandrosterone sulfate (DHEAS), cortisol anddehydroepiandrosterone (DHEA) Polycystic ovary syndrome: testosteroneAndropause: testosterone; testosterone precursors such as pregnenolone,progesterone, 17- hydroxypregnenolone, 17-hydroxyprogesterone,dehydroepiandrosterone (DHEA) and delta-4-androstene-3,17-dione;testosterone and dihydrotestosterone metabolites such as the17-ketosteroids androsterone and etiochol anol one, polar metabolites inthe form of diols, triols, and conjugates, as well as estradiol,estrogens, androsteindione, cortisol, FSH (follicle stimulatinghormone), LH (luteinizing hormone), and GnRH (gonadotropin- releasinghormone) Coagulation status/disorders: b-Thromboglobulin, Plateletfactor 4, Von Willebrand factor, Factor I: Fibrinogen, Factor II:Prothrombin, Factor III: Tissue factor, Factor IV: Calcium, Factor V:Proaccelerin, Factor VI, Factor VII: Proconvertin, Factor VIII:, Anti-hemolytic factor, Factor IX: Christmas factor, Factor X: Stuart-Prowerfactor, Factor XI: Plasma thromboplastin antecedent, Factor XII: Hagemanfactor, Factor XIII: Fibrin- stabilizing factor, Prekallikrein,High-molecular-weight kininogen, Protein C, Protein S, D- dimer, Tissueplasminogen activator, Plasminogen, a2-Antiplasmin, Plasminogenactivator inhibitor 1 (PAI1) Autism: miR-484, miR-21, miR-212, miR-23a,miR-598, miR-95, miR-129, miR-431, miR-7, miR-15a, miR-27a, miR-15b,miR-148b, miR-132, or miR-128; miR-93, miR- 106a, miR-539, miR-652,miR-550, miR-432, miR-193b, miR-181d, miR-146b, miR-140, miR-381,miR-320a, or miR-106b; GM1, GD1a, GD1b, orGT1b; Ceruloplasmin,Metalothioneine, Zinc, Copper, B6, B12, Glutathione, Alkalinephosphatase, and Activation of apo-alkaline phosphatases Alzheimer’sDisease: miR-107, miR-29a, miR-29b-1, or miR-9; miR-128; HIF-1α, BACE1,Reelin, CHRNA7, or 3Rtau/4Rtau, Reelin, Cystatin C, Truncated CystatinC, C3a, t-Tau, Complement factor H, or alpha-2-macroglobulin;β-amyloid(1-42), β- amyloid(1-40), tau, phosphor-tau-181,acetylcholinesterase enzyme (AChE), GSK-3, PKC, VCAM-1 and ICAM-1,macrophage inflammatory proteins-1δ and -4 (MIP1δ and MIP4), regulatedupon activation normal T-cell (RANTES), tumor necrosis factor-alpha(TNFα), midregional pro-atrial natriuretic peptide (MR-proANP),AD-associated neuronal thread protein (AD7c-NTP) Parkinson’s Disease:miR-133b; Nurr1, BDNF, TrkB, gstm1, or 5100 beta; apo-H, Ceruloplasmin,BDNF, Beta2-microglobulin, apoAII, tau, ABeta1-42, DJ-1, cTnI,myoglobin, MMP-9, MMP-8, MMP-2, sICAM-1, myeloperoxidase [MPO], IL-4,and/or IL-5; B-type natiuretic peptide [BNP], IL-1α, IL-11, IL-10,TNF-α, IFN-γ, VEGF, insulin, GLP-1 (active), GLP-1 (total), TREM1,Leukotriene E4, Akt1, Aβ-40, Aβ-42, Fas ligand, PSA, G-CSF, MIP-1α,IL-22, IL-8, IL-21, IL-15, IL-6, IL-7, GM-CSF, IL-2, IL-12, IL- 17α,IL-1β, MCP, IL-32 or RANTES, apolipoproteins A1, D and E,ischemia-modified albumin (IMA), fibronectin, s. alpha-amylase,aspartate aminotransferase, lactate dehydrogenase, tissue factoractivity, MCP-1, sVCAM-1, sCD-40, insulin-like growth factor I (IGF-I),IGF-II Schizophrenia: miR-181b; miR-7, miR-24, miR-26b, miR-29b,miR-30b, miR-30e, miR- 92, ormiR-195; IFITM3, SERPINA3, GLS, orALDH7A1BASP1; TP5B, ATP5H, ATP6V1B, DNM1, NDUFV2, NSF, PDHB Bipolardisease: FGF2, ALDH7A1, AGXT2L1, AQP4, or PCNT2 Mood disorder: Mbp,Edg2, Fgfr1, Fzd3, Mag, Pmp22, Ugt8, Erbb3, Igfbp4, Igfbp6, Pde6d,Ptprm, Nefh, Atp2c1, Atxn1, Btg1, C6orf182, Dicer1, Dnajc6, and EdnrbMajor Depressive Disorder: FGFR1, FGFR2, FGFR3, or AQP4, Secretogranin,VGF, Cortisol, EGF, GCS, PPY, ACTH, AVP, CRH, AIAT, A2M, ApoC3, CD40L,IL-6, IL-13, IL-18, IL-1ra, MPO, PAI-1, TNFA, ACRP30, ASP, FABP, INS,LEP, PRL, RETN, Testosterone, TSH, BDNF, S100B, NTF3, GDNF, ARTN Priondisease: Amyloid B4, App, IL-1R1, or SOD1; PrP(c), 14-3-3, NSE, S-100,Tau, AQP-4 Inflammation: TNF-α, IL-6, IL1β, Rheumatoid factor (RF),Antinuclear Antibody (ANA), acute phase markers including C-reactiveprotein (CRP), Clara Cell Protein (Uteroglobin); 14-3-3 protein epsilon;Isoform Long of Protocadherin alpha C2 precursor; Insulin-like growthfactor IA precursor; Isoform 1 of Protocadherin-8 precursor; Isoform 1of Sodium/potassium/calcium exchanger 2 precursor; Complement factorH-related 5; Di-N- acetylchitobiase precursor; Isoform 1 of ProteinNDRG2; N-acetylglucosamine-6-sulfatase precursor; Isoform 1 ofSemaphorin-3B precursor; Cadherin-5 precursor; UPF0454 protein C12orf49precursor; Dihydrolipoyl dehydrogenase, mitochondrial precursor;Metallothionein-3; Fas apoptotic inhibitory molecule 2; Coactosin-likeprotein; Isoform Long of Platelet-derived growth factor A chainPrecursor; Isoform Long of Endothelin-3 precursor; HLA class Ihistocompatibility antigen, A-1 alpha chain Precursor; Neuronalpentraxin-2 precursor; retbindin isoform 2; Neuroendocrine convertase 2precursor; 15 kDa selenoprotein isoform 1 precursor; Phospholipase D4;Isoform 1 of CD109 antigen precursor; Ectonucleotidepyrophosphatase/phosphodiesterase family; member 6 precursor; Fascin;Golgi phosphoprotein 2; Isoform Delta 6 of Calcium/calmodulin-dependentprotein kinase type II delta chain; Isoform 1 of FRASI-relatedextracellular matrix protein 2 Precursor; Putative uncharacterizedprotein LOC130576; Isoform 1 of L-lactate dehydrogenase A chain; Isoform1 of Polypeptide N-acetylgalactosaminyltransferase 13; Papilin; ProteinDJ-1; Beta-mannosidase precursor; Protein YIPF3; Isoform 1 of Receptor-type tyrosine-protein phosphatase N2 Precursor; Cell growth regulatorwith EF hand domain protein 1; Sulfhydryl oxidase 2 precursor; Ig lambdachain V-II region TRO; Ig lambda chain V-VI region AR; Ig heavy chainV-III region WEA; Ig heavy chain V-III region CAM; Ig heavy chain V-IIIregion BUR; Myosin-reactive immunoglobulin kappa chain variable region(Fragment); Microfibrillar protein 2 (Fragment); Ig kappa chain V-IIIregion IARC/BL41 precursor; Ig kappa chain V-I region Kue; Ig kappachain V-I region Scw; Ig kappa chain V-III region B6; IGLV6-57 protein;hypothetical protein LOC402665; Isoform 1 of Proline-rich acidic protein1 precursor; Rheumatoid factor RF-ET13; Rheumatoid factor D5 heavy chain(Fragment); Uncharacterized protein ENSP00000375027; Uncharacterizedprotein ENSP00000375043; Uncharacterized protein ENSP00000375019;Isoform 1 of Protocadherin-1 precursor; Isoform 1 of Epithelialdiscoidin domain-containing receptor 1 precursor; Serine protease HTRA1precursor; Isoform Delta of Poliovirus receptor-related protein lPrecursor; chemokine (C X C motif) ligand 16; Plastin-2; 14-3-3 proteinzeta/delta; Apolipoprotein C-II precursor; Brain- specific angiogenesisinhibitor 1 precursor; Semaphorin-3G precursor; Follistatin-relatedprotein 3 precursor; Hepatocyte growth factor activator precursor;Isoform 1 of Contactin- associated protein-like 2 precursor;Phosphoglycerate kinase 1; Gamma-enolase; Phosphoglycerate mutase 2; Lowaffinity immunoglobulin gamma Fc region receptor III-A precursor;Isoform Beta of Poliovirus receptor precursor; Serine protease inhibitorKazal- type 6 precursor; Isoform 1 of Chordin precursor; Out at firstprotein homolog precursor; Isoform 1 of Carboxypeptidase B2 precursor;ROBO2 isoform a ig kappa chain V-III region POM; Isoform 1 ofProtein-L-isoaspartate(D-aspartate) O-Methyltransferase CDNA FLJ45296fis, clone BRHIP3003340, moderately similar to Actin, alpha skeletalmuscle 2; Isoform 1 of RGM domain family member B precursor;Carboxypeptidase N subunit 2 precursor; Hypothetical LOC284297; L-6,IL-17, PAR-3, IL-17, T1/ST2, JunD, 5-LO, LTA4H, MBP, PLP, or alpha-betacrystalline; antithrombin III; α-2 glycoprotein 1, zinc; transthyretin(prealbumin); NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 2;neurotrimin; orosomucoid 1 precursor (α-1-acid glycoprotein-1);leucine-rich α-2- glycoprotein; leucine-rich repeat protein;α-1-antitrypsin Chronique fatigue syndrome: Cortisol; Ig alpha-1 chain Cregion; Polymeric immunoglobulin receptor; Protein S100-A7; Cystatin-C;Cystatin-B; 14-3-3 protein zeta/delta; Zinc-alpha-2-glycoprotein (ZAG)Sjogren’s syndrome: IgA, IgG, IgM autoantibodies; IgA, lactoferrin andbeta2- microglobulin; lysozyme C, and cystatin C, amylase and carbonicanhydrase; Autoantibodies (SSA/Ro; LA/SS-B) Systemic lupus erythematosus(SLE): Autoantibodies (CDC25B, APOBEC3G, ARAF, BCL2A1, CLKI, CREB1,CSNK1G1, CSNK2A1, CWC27, DLX4, DPPA2, EFHD2, EGR2, ERCC2, EWSR1, EZH2,FES, FOS, FTHL17, GEM, GNA15, GNG4, HMGB2, HNRNPULl, HOXB6, ID2, IFI35,IGF2BP3, IGHG1, JUNB, KLF6, LGALS7, LIN28A, MLLT3, NFIL3, NRBF2, PABPC1,PATZI, PCGF2, PPP2CB, PPP3CC, PRM1, PTK2, PTPN4, PYGB, RET, RPL18A,RPS7, RRAS, SCEL, SH2B1, SMAD2, STAM, TAF9, TIE1, UBA3, VAV1, WT1,ZAP70, ZNRD1, KIT, C6orf93, RPL34, DOM3Z, COPG2, DNCL12, RRP41, FBXO9,RALBP1, PIAS2, EEF1D, CONI, KATNB1, POLR2E, CCT3, KIAA0643, RPL37A,GTF2H2, MAP2K5, CDK3, RPS6KA1, MARK4, MTO1, MGC42105, NFE2L2, WDR45L,STK4, PFKFB3, NTRK3, MLF1, TRIM37, ACTL7B, RPL18A, CKS1B, TUBA1, NME6,SUCLA2, IGHG1, PRKCBP1, BAG3, TCEB3, RPL15, SSX4, MAP2K7, EEF1G, RNF38,PHLDA2, KCMF1, NUBP2, VPS45A, SSA/Ro, dsDNA, Smith, histones, thrombin)CREST syndrome: Autoantibodies (centromere) Systemic sclerosis:Autoantibodies (Type I topoisomerase) Primary biliary cirrhosis:Autoantibodies (nucleoporin 62, Sp100 nuclear antigen, nucleoporin210kDa, mitochondria) Cirrhosis: NLT; NLT, HBsAG, AST, YKL-40,Hyaluronic acid, TIMP-1, alpha 2 macroglobulin, a-1-antitrypsin P1Zallele, haptoglobin, or acid phosphatase ACP AC Autoimmune hepatitis:Autoantibodies (Liver kidney microsomal type 1, smooth muscle) Celiacdisease: Autoantibodies (tTG, actin) Celiac disease Irritable BowelSyndrome (IBS): Anti-IgA gliadin, REGIA, MMP3 Inflammatory bowel disease(IBD): Trypsinogen IV, SERT; II-16, II-lbeta, II-12, TNF- alpha,interferon gamma, 11-6, Rantes, MCP-1, Resistin, or 5-HT Ulcerativecolitis: IFITM1, IFITM3, STAT1, STAT3, TAPI, PSME2, PSMB8, HNF4G, KLF5,AQP8, APT2B1, SLC16A, MFAP4, CCNG2, SLC44A4, DDAH1, TOB1, 231152_at,MKNK1, CEACAM7*, 1562836_at, CDC42SE2, PSD3, 231169_at, IGL@*, GSN,GPM6B, CDV3*, PDPK1, ANP32E, ADAM9, CDH1, NLRP2, 215777_at, OSBPL1,VNN1, RABGAP1L, PHACTR2, ASH1L, 213710_s_at, CDH1, NLRP2, 215777_at,OSBPL1, VNN1, RABGAP1L, PHACTR2, ASH1, 213710_s_at, ZNF3, FUT2, IGHA1,EDEM1, GPR171, 229713_at, LOC643187, FLVCR1, SNAP23*, ETNK1, LOC728411,POSTN, MUC12, HOXA5, SIGLEC1, LARP5, PIGR, SPTBN1, UFM1, C6orf62, WDR90,ALDH1A3, F2RL1, IGHV1-69, DUOX2, RAB5A, or CP; (P)ASCA HyperplasticPolyp: SLC6A14, ARHGEF10, ALS2, IL1RN, SPRy4, PTGER3, TRIM29, SERPINB5,1560327 at, ZAK, BAG4, TRIB3, TTL, FOXQ1 Psoriasis: miR-146b, miR-20a,miR-146a, miR-31, miR-200a, miR-17-5p, miR-30e-5p, miR-141, miR-203,miR-142-3p, miR-21, or miR-106a; miR-125b, miR-99b, miR-122a, miR-197,miR-100, miR-381, miR-518b, miR-524, let-7e, miR-30c, miR-365, miR-133b,miR-10a, miR-133a, miR-22, miR-326, or miR-215; IL-20, VEGFR-1, VEGFR-2,VEGFR-3, orEGR1; Dermatitis herpetiformis: Autoantibodies (eTG)Miller-Fisher Syndrome: Autoantibodies (ganglioside GQ1B) Wegener'sgranulomatosis: Autoantibodies (c-ANCA) Neuropathies: Autoantibodies(ganglioside GD3, ganglioside GM1) Microscopic polyangiitis:Autoantibodies (p-ANCA) Polymyositis: Autoantibodies (Signal recognitionparticles) Scleromyositis: Autoantibodies (exosome complex Signalrecognition particles) Myasthenia gravis: Autoantibodies (nicotinicacetylcholine receptor Signal recognition particles, muscle-specifickinase (MUSK) Signal recognition particles) Lambert-Eaton myasthenicsyndrome: Autoantibodies (voltage-gated calcium channel (P/Q-type))Hashimoto's thyroiditis: Autoantibodies (thyroid peroxidase) Graves'disease: Autoantibodies (TSH receptor) Paraneoplastic cerebellarsyndrome: Autoantibodies (Hu, Yo (cerebellar Purkinje Cells),amphiphysin) Encephalitis: Autoantibodies (voltage-gated potassiumchannel (VGKC), N-methyl-D- aspartate receptor (NMDA)) Sydenham'schorea: Autoantibodies (basal ganglia neurons) Neuromyelitis:Autoantibodies (aquaporin-4) Allergies: Allergen-specific IgAs Rheumaticdisease: miR-146a, miR-155, miR-132, miR-16, or miR-181; HOXD10, HOXD11,HOXD13, CCL8, LIM homeobox2, or CENP-E; TNFa Rheumatoid arthritis:Autoantibodies (Rheumatoid factor, cyclic citrullinated protein),ATP-binding cassette, sub-family A, member 12 isoform b; ATP-bindingcassette A12; apolipoprotein; B-100 precursor - human; complementcomponent 3 precursor; alpha-2- glycoprotein l,zinc;Alpha-2-glycoprotein, zinc; serine (or cysteine) proteinase inhibitor,clade A (alpha-1 antiproteinase, antitrypsin), member 2; Proteaseinhibitor 1-like; protease inhibitor 1 (alpha-l-antitrypsin)-like;group-specific component (vitamin D binding protein); hDBP; serine (orcysteine) proteinase inhibitor, clade A (alpha-1 antiproteinase,antitrypsin), member 1; Protease inhibitor (alpha-1-antitrypsin);protease inhibitor 1 (anti- elastase), alpha-1-antitrypsin; Vitronectinprecursor V65 subunit; A kinase anchor protein 9 isoform 2;retrovirus-related hypothetical protein II -human retrotransposonLINE-1; nuclear receptor coactivator RAP250; peroxisomeproliferator-act; nuclear receptor coactivator RAP2; Ig kappa chainNIG26 precursor - human; Vitamin D-binding protein precursor (DBF)(Group-specific component) (GC-globulin) (VDB) complement C4A precursor[validated] Human; guanine nucleotide binding protein (G protein), gammatransducing activity polypeptide 1; nucleoporin 98kD isoform 4;nucleoporin 98kD; Nup98-Nup96 precursor; GLFG-repeat containing;nucleoporin; vitronectin precursor; serum spreading factor; somatomedinB; complement S-protein; Alpha-1-antitrypsin precursor; HMG-BOXtranscription; factor BBX; x 001; protein; hect domain and RLD 2;calcium channel, voltage-dependent, L type, alpha IC subunit;Alpha-2-antiplasmin precursor (Alpha-2-plasmin inhibitor) (Alpha-2-PI)(Alpha-2-AP); Neuronal PAS domain protein 2 (Neuronal PAS2) (Member ofPAS protein 4) (MOP4); Retinoic acid receptor gamma-2 (RAR-gamma-2)alpha-1-B-glycoprotein - human; Heparin cofactor II precursor (HC-II)(Protease inhibitor leuserpin 2) (HLS2); Ig gamma-1 chain C region;isocitrate dehydrogenase 3 (NAD+) alpha precursor; H-IDH alpha;isocitric dehydrogenase; isocitrate dehydrogenase [NAD] sub- unit alpha,mitochondrial; NAD+-specific ICDH; NAD(H)-specific isocitratedehydrogenase alpha subunit precursor; isocitrate dehydrogenase (NAD+)alpha chain precursor; ferroxidase (EC 1.16.3.1) precursor [validated] -human; similar to zona pellucida binding protein; N-acetylneuraminicacid phosphate synthase; sialic acid synthase; sialic acid phosphatesynthase; triple functional domain (PTPRF interacting); deleted inbladder cancer chromosome region candidate 1; ceruloplasmin(ferroxidase); Ceruloplasmin; RAB3A interacting protein (rabin3)-like 1;talin 2; similar to Ceruloplasmin precursor (Ferroxidase); orosomucoid 1precursor; Orosomucoid-1 (alpha-1-acid glycoprotein-1); Ig lambda chainprecursor - human; cold autoinflammatory syndrome 1; chromosome 1 openreading frame 7; angio- tensin/vasopressin receptor; similar to KIAA0913protein; sodium channel, voltage-gated, type V, alpha polypeptide;hypothetical protein FLJ10379; orosomucoid 2; alpha-1-acid glycoprotein,type 2; Ig alpha-1 chain C region; corticosteroid binding globulinprecursor; corticosteroid binding globulin; alpha-1 anti-proteinase,antitrypsin; KV3M_HUMAN IG KAPPA CHAIN V-III REGION HIC PRECURSOR;MUC_HUMAN Ig mu chain C region; similar to Ig gamma-2 chain C region;alpha-1-antichymotrypsin, precursor; alpha- 1-anti chymotrypsin; Antichymotrypsin; thyroid hormone receptor-associated protein, 240 kDasubunit; Ig heavy chain - human; Alpha-1-antichymotrypsin precursor(ACT) hypothetical protein XP_173158; hypothetical proteinDKFZp434G2226; haptoglobin; Plasma protease Cl inhibitor precursor (ClInh) (Clinh) Haptoglobin-1 precursor; leucine- richalpha-2-glycoprotein; S-arrestin; S-antigen; NAD(P)H dehydrogenase,quinone 2; NAD(P)H menadione oxidoreductase-1, di-oxin-inducible-2;NAD(P)H menadione oxi- doreductase 2, dioxin-inducible; angiotensinprecursor [validated] - human; similar to KIAA1902 protein; similar toKIAA1728 protein; calpain 3 isoform d; calpain, large polypep- tide L3;calpain p94, large [catalytic] subunit; muscle-specificcalcium-activated neutral protease 3 large subunit; asp (abnormalspindle)-like, microcephaly associated; haptoglobin-related protein;Haptoglobin-related locus; Ig alpha-2 chain C region; hypotheticalprotein DKFZp434P1818.1 - human (fragment); GC3_HUMAN Ig gamma-3 chain Cregion (Heavy chain disease protein) (HDC) Organ Rejection: miR-658,miR-125a, miR-320, miR-381, miR-628, miR-602, miR-629, or miR-125a;miR-324-3p, miR-611, miR-654, miR-330_MM1, miR-524, miR-17- 3p_MM1,miR-483, miR-663, miR-5,6-5p, miR-326, miR-197_MM2, or miR-346; matixmetalloprotein-9, proteinase 3, orHNP Bone turnover/ Osteoporosis:Pyridinoline, deoxypyridinoline, collagen type 1 corss- linkedN-telopeptide (NTX), collagen type 1 corss-linked C-telopeptide (CTX),bone sialoprotein (BSP), Tartrate-resistant acid phosphatase 5b,deoxypyridinium (D-PYR) and osteocalcin (OC), hepatocyte growth factorand interleukin-1 beta, Osteocalcin, alkaline phosphatase, bone-specificalkaline phosphatase, serum type 1 procollagen (CINP, P1NP) Jawosteonecrosis: PTH, insulin, TNF-α, leptin, OPN, OC, OPG and IL6Gaucher’s disease: lyso-Gbl, Chitotriosidase and CCL18 Traumatic braininjury: apoA-1, S-100B, isoprostane, GFAP, NGAL, neuron-specific enolase(NSE) Septic shock: 15-Hydroxy-PG dehydrogenase (up), LAIR1 (up), NFKB1A(up), TLR2, PGLYPR1, TLR4, MD2, TLR5, IFNAR2, IRAK2, IRAK3, IRAK4, PI3K,PI3KCB, MAP2K6, MAPK14, NFKB1A, NFKB1, IL1R1, MAP2K1IP1, MKNK1, FAS,CASP4, GADD45B, SOCS3, TNFSF10, TNFSF13B, OSM, HGF, IL18R1, IL-6,Protein-C, IL- 1beta Cancer: FEN-1; CEA, NSE, CA 19-9, CA 125, PSA,proGRP, SCC, NNMT, anti-p53 autoantibodies, Separase and DPPFV/Separase,SERPINA3; ACTB; AFM; AGT; AMBP; APOF; APOA2; APOC1; APOE; APOH;SERPINC1; Cl QB; C3; C4BPA; C8G; C9; SERPINA6; CD14; CP; CRP; CSK; F9;FGA; FGG; FLNA; FN1; GC; HRG; IF; IGFALS; ITGA1; ITIH1; ITIH2; ITIH4;KLKB1; LPA; MLL; MRC1; MYL2; MYO6; ORM1; SERPINF1; SERPINA1; SERPINA4;PROS1; QSCN6; RGS4; SAA4; SERPINA7; TF; TFRC; TTN; UBC; ALMS1; ATRN;PDCD11; KIAA0433; SERPINA10; BCOR; C10orf18; YY1AP1; FLJ10006; BDP1;SMARCAD1; MKL2; CHST8; MCPH1; MYO18B; MICAL-L1; PGLYRP2; KCTD7;MGC27165; A1BG; A2M; ABLIMI; ACTA1; AHSG; ANK3; APCS; APOA1; APOA4;APOB; APOC3; APOL1; AZGP1; B2M; BF; C1R; CIS; C2; C4B; C5; C6; C7; C8A;C8B; CDK5RAP2/CDK5RA2; CHGB; CLU; COMP; COROIA; CPN1; CULl; DET1; DSC1;F13A1; F2; F5; FGB; GOLGA1; GSN; HBA1; HBB; HP; HPX; HSPA5; HUNK;IGFBP5; IGHGI; IGLV4-3; KIF5C; KNG1; KRT1; KRT10; KRT9; LBP; LGALS3BP;LRG1; LUM; MMP14; MYH4; NEB; NUCB2; ORM2; PF4V1; PIGR; PLG; PON1; PPBP;RBP4; RIMS1; RNF6; SAA1; SEMA3D; SERPIND1; SERPINF2; SERPING1; SF3B1;SPINK1; SPP1; SPTB; SYNE1; TAF4B; TBC1D1; TLN1; TMSB4X; TRIP11; TTR;UROC1; VTN; VWF; ZFHX2; ZYX; PSA (total prostate specific antigen),Creatinine, Prostatic acid phosphatase, PSA complexes,Prostrate-specific gene-1, CA 12- 5, Carcinoembryonic Antigen (CEA),Alpha feto protein (AFP), hCG (Human chorionic gonadotropin), Inhibin,CAA Ovarian C1824, CA 27.29, CA 15-3, CAA Breast C1924, Her-2,Pancreatic, CA 19-9, CAA pancreatic, Neuron-specific enolase,Angiostatin DcR3 (Soluble decoy receptor 3), Endostatin, Ep-CAM (MK-1),Free Immunoglobulin Light Chain Kappa, Free Immunoglobulin Light ChainLambda, Herstatin, Chromogranin A, Adrenomedullin, Integrin, Epidermalgrowth factor receptor, Epidermal growth factor receptor-Tyrosinekinase, Pro-adrenomedullin N-terminal 20 peptide, Vascular endothelialgrowth factor, Vascular endothelial growth factor receptor, Stem cellfactor receptor, c- kit/KDR, KDR, and Midkine; Zinc α2 -glycoprotein(ZAG) Adenoma: SI, DMBT1, CFI*, AQP1, APOD, TNFRSF17, CXCL10, CTSE,IGHA1, SLC9A3, SLC7A1, BATF2, SOCS1, DOCK2, NOS2A, HK2, CXCL2, IL15RA,POU2AF1, CLEC3B, ANI3BP, MGC13057, LCK*, C4BPA, HOXC6, GOLT1A, C2orf32,IL10RA, 240856_at, SOCS3, MEIS3P1, HIPK1, GLS, CPLX1, 236045_x_at, GALC,AMN, CCDC69, CCL28, CPA3, TRIB2, HMGA2, PLCL2, NR3C1, EIF5A, LARP4,RP5-1022P6.2, PHLDB2, FKBP1B, INDO, CLDN8, CNTN3, PBEF1, SLC16A9,CDC25B, TPSB2, PBEF1, ID4, GJB5, CHN2, LIMCH1, or CXCL9; ABCA8,KIAA1199, GCG, MAMDC2, C2orf32, 229670_at, IGF1, PCDH7, PRDX6, PCNA,COX2, or MUC6 Head and Neck cancer: IL-1, IL-6, IL-8, VEGF, MMP-9,TGF-β, TNF-α, MMP-7, plasminogen activated (PA), uPA, IGF, or INF-2Barrett’s esophagus: miR-21, miR-143, miR-145, miR-194, or miR-215;S100A2, S100A4; p53, MUC1, MUC2 Lung cancer: miR-21, miR-205, miR-221(protective), let-7a (protective), miR-137 (risky), miR-372 (risky), ormiR-122a (risky); miR-17-92, miR-19a, miR-92, miR-155, miR-191, ormiR-210; EGFR, PTEN, RRM1, RRM2, ABCB1, ABCG2, LRP, VEGFR2, VEGFR3,class III b-tubulin; KRAS, hENTI; RLF-MYCL1, TGF-ALK, or CD74-ROS1,CCNI, EGFR, FGF19, FRS2, and GREB1 LZTS, BRAF, FRS2, ANXA1, HaptoglobinHp2, Zinc Alpha2-Glycoprotein, Calprotectin, Porphyromonas catoniae 16SrRNA, Campylobacter showae 16S rRNA, Streptocococcus salivaris 16S rRNA,Campylobacter rectus 16S rRNA, Veillonella parvula 16S rRNA, Kigellaoralis 16S rRNA, and Granulicatella adiacens 16S rRNA Pancreatic cancer:miR-221, miR-181a, miR-155, miR-210, miR-213, miR-181b, miR- 222,miR-181b-2, miR-21, miR-181b-l, miR-220, miR-181d, miR-223, miR-100-1/2,miR-125a, miR-143, miR-10a, miR-146, miR-99, miR-100, miR-199a-1,miR-10b, miR- 199a-2, miR-221, miR-181a, miR-155, miR-210, miR-213,miR-181b, miR-222, miR- 181b-2, miR-21, miR-181b-1, miR-181c, miR-220,miR-181d, miR-223, miR-100-1/2, miR-125a, miR-143, miR-10a, miR-146,miR-99, miR-100, miR-199a-l, miR-10b, miR- 199a-2, miR-107, miR-103,miR-103-2, miR-125b-l, miR-205, miR-23a, miR-221, miR- 424, miR-301,miR-100, miR-376a, miR-125b-l, miR-21, miR-16-1, miR-181a, miR- 181c,miR-92, miR-15, miR-155, let-7f-l, miR-212, miR-107, miR-024-1/2,miR-18a, miR-31, miR-93, miR-224, orlet-7d; miR-148a, miR-148b, miR-375,miR-345, miR-142, miR-133a, miR-216, miR-217 or miR-139; KRAS, CTNNLB1,AKT, NCOA3, or B-RAF; BRCA2, PALB2, or p16, MBD3L2, KRAS, STIM2, DMXL2,ACRV1, DMD and CABLES1, TK2, GLTSCR2, CDKL3, TPT1 and DPM1 Breastcancer: miR-21, miR-155, miR-206, miR-122a, miR-210, miR-155, miR-206,miR-210, or miR-21; let-7, miR-10b, miR-125a, miR-125b, miR-145,miR-143, miR-16, miR-10b, miR-125a; hsp70, MART-1, TRP, HER2, hsp70,MART-1, TRP, HER2, ER, PR, Class III b-tubulin, or VEGF A; GAS5;ETV6-NTRK3; CAH6 (Carbonic anhydrase VI), K2C4 (Cytokeratin 4), CYTA(Cystatin A), FABP4 (Epid. Fatty acid binding prot.), IGHGI (Ig gamma-1chain C region), TRFL (Lactoferrin), BPIL1 (Bact. Perm.-increasingprot.-1), CYTC (Cystatin C), HPT (Haptoglobin), PROF1 (Profilin-1), ZA2G(Zinc-alpha- 2-glycoprotein), ENOA (Alpha enolase), IGHA2 (1g alpha-2chain C region), IL-1 ra (Interleukin-1 receptor anatagonist proteinprecursor), S10A7 (S100 calcium-binding protein A7), and SPLC2 (Shortpalate, lung and nasel epith Care, assoc, protein 2) Ovarian cancer:c-erbB-2, cancer antigen 15-3, p53, HER2/neu (c-erbB-2), 47D10 antigen,PTCD2, SLC25A20, NFKB2, RASGRP2, PDE7A, MLL, PRKCE, GPATC3, PRIC285 andGSTA4, MIPEP, PLCB2, SLC25A19, DEF6, ZNF236, C18orf22, COX7A2, DDX11,TOP3A, C9orf6, UFC1, PFDN2, KLRD1, LOC643641, HSP90AB1, CLCN7, TNFAIP2,PRKCE, MRPL40, FBF1, ANKRD44, CCT5, USP40, UBXD4, LRCH1, MRPL4, SCCPDH,STX6, LOC284184, FLJ23235, GPATC3, CPSF4, CREM, HISTIHID, HPS4, FN3KRP,ANKRD16, C8 orf16, ATF71P2, PRIC285, miR-200a, miR- 141, miR-200c,miR-200b, miR-21, miR-200a, miR-200b, miR-200c, miR-203, miR-205,miR-214, miR-199″, or miR-215; miR-199a, miR-140, miR-145, miR-100,miR-let-7 cluster, or miR-125b-1; ERCC1, ER, TOPO1, TOP2A, AR, PTEN,CD24 or EGFR; VEGFA, VEGFR2, CA 125 Prostate cancer: AGPAT1, B2M, BASP2,IER3, IL1B, miR-9, miR-21, miR-141, miR- 370, miR-200b, miR-210,miR-155, or miR-196a; miR-202, miR-210, miR-296, miR-320, miR-370,miR-373, miR-498, miR-503, miR-184, miR-198, miR-302c, miR-345, miR-491, miR-513, miR-32, miR-182, miR-31, miR-26a-l/2, miR-200c, miR-375,miR-196a- 1/2, miR-370, miR-425, miR-425, miR-194-1/2, miR-181a-l/2,miR-34b, let-71, miR-188, miR-25, miR-106b, miR-449, miR-99b, miR-93,miR-92-1/2, miR-125a, or miR-141; let- 7a, let-7b, let-7c, let-7d,let-7g, miR-16, miR-23a, miR-23b, miR-26a, miR-92, miR-99a, miR-103,miR-125a, miR-125b, miR-143, miR-145, miR-195, miR-199, miR-221, miR-222, miR-497, let-7f, miR-19b, miR-22, miR-26b, miR-27a, miR-27b,miR-29a, miR-29b, miR-30_5p, miR-30c, miR-100, miR-141, miR-148a,miR-205, miR-520h, miR-494, miR- 490, miR-133a-l, miR-1-2, miR-218-2,miR-220, miR-128a, miR-221, miR-499, miR-329, miR-340, miR-345, miR-410,miR-126, miR-205, miR-7-1/2, miR-145, miR-34a, miR- 487, or let-7b;miR-15a, miR-16-1, miR-143 or miR-145; AR, PCA3; FASLG or TNFSF10; U50;ACSL3-ETV1, C15ORF21-ETV1, FLJ35294-ETV1, HERV-ETV1, TMPRSS2-ERG,TMPRSS2-ETV1/4/5, TMPRSS2-ETV4/5, SLC5A3-ERG, SLC5A3- ETV1, SLC5A3-ETV5,KLK2-ETV4, kallikrein-2 (KLK2), C reactive protein (CRP), cysteine-richsecretory protein 3 (CRISP3) and chromogranin A (CHGA), comprisesprostatic acid phosphatase (PAP), lactate dehydrogenase (LDH), alkalinephosphatase (ALP), PSA Esophageal Cancer: PCA3, GOLPH2, SPINK 1,TMPRSS2:ERG, miR-192, miR-194, miR-21, miR-200c, miR-93, miR-342,miR-152, miR-93, miR-25, miR-424, or miR-151; miR-27b, miR-205, miR-203,miR-342, let-7c, miR-125b, miR-100, miR-152, miR-192, miR-194, miR-27b,miR-205, miR-203, miR-200c, miR-99a, miR-29c, miR-140, miR-103, miR-107Gastric cancer: miR-106a, miR-21, miR-191, miR-223, miR-24-1, miR-24-2,miR-107, miR-92-2, miR-214, miR-25, or miR-221; let-7a; RRM2, orsurviving; EphA4 Gastrointestinal Stromal Tumor (GIST): DOG-1,PKC-theta, KIT, GPR20, PRKCQ, KCNK3, KCNH2, SCG2, TNFRSF6B, or CD34;PDGFRA, c-kit Colorectal carcinoma: miR-24-1, miR-29b-2, miR-20a,miR-10a, miR-32, miR-203, miR-106a, miR-17-5p, miR-30c, miR-223,miR-126, miR-128b, miR-21, miR-24-2, miR- 99b, miR-155, miR-213,miR-150, miR-107, miR-191, miR-221, miR-20a, miR-510, miR- 92, miR-513,miR-19a, miR-21, miR-20, miR-183, miR-96, miR-135b, miR-31, miR-21,miR-92, miR-222, miR-181b, miR-210, miR-20a, miR-106a, miR-93, miR-335,miR-338, miR-133b, miR-346, miR-106b, miR-153a, miR-219, miR-34a,miR-99b, miR-185, miR- 223, miR-211, miR-135a, miR-127, miR-203,miR-212, miR-95, or miR-17-5p; miR-143, miR-145, miR-143, miR-126,miR-34b, miR-34c, let-7, miR-9-3, miR-34a, miR-145, miR- 455, miR-484,miR-101, miR-145, miR-133b, miR-129, miR-124a, miR-30-3p, miR-328,miR-106a, miR-17-5p, miR-342, miR-192, miR-1, miR-34b, miR-215, miR-192,miR-301, miR-324-5p, miR-30a-3p, miR-34c, miR-331, or miR-148b; EFNB1,ERCC1, HER2, VEGF, or EGFR; AFRs, Rabs, ADAMI0, CD44, NG2, ephrin-B1,MIF, b-catenin, Junction, plakoglobin, glalectin-4, RACK1, tetrspanin-8,FasL, TRAIL, A33, CEA, EGFR, dipeptidase 1, hsc-70, tetraspanins, ESCRT,TS, PTEN, or TOPO1; GREM1, DDR2, GUCY1A3, TNS1, ADAMTSI, FBLN1,FLJ38028, RDX, FAM129A, ASPN, FRMD6, MCC, RBMS1, SNA12, MEISI, DOCK10,PLEKHC1, FAM126A, TBC1D9, VWF, DCN, ROBO1, MSRB3, LATS2, MEF2C, IGFBP3,GNB4, RCN3, AKAP12, RFTN1, 226834_at, COL5A1, GNG2, NR3C1*, SPARCL1,MAB21L2, AXIN2, 236894_at, AEBP1, AP1S2, C10orf56, LPHN2, AKT3, FRMD6,COL15A1, CRYAB, COL14A1, LOC286167, QKI, WWTR1, GNG11, PAPPA, orELDTl;227458_at, INDO, CXCL9, CCR2, CD38, RARRES3, CXCL10, FAM26F, TNIP3,NOS2A, CCRL1, TLR8, IL18BP, FCRL5, SAMD9L, ECGF1, TNFSF13B, GBPS, orGBP1; TMEM37*, IL33, CA4, CCDC58, CLIC6, VERSUSNL1, ESPN, APCDD1,C13orfl8, CYP4X1, ATP2A3, LOC646627, MUPCDH, ANPEP, Clorf115, HSD3B2,GBA3, GABRB2, GYLTL1B, LYZ, SPC25, CDKN2B, FAM89A, MOGAT2, SEMA6D,229376_at, TSPAN5, IL6R, or SLC26A2 Melanoma: miR-19a, miR-144,miR-200c, miR-211, miR-324-5p, miR-331, or miR-374; miR-9, miR-15a,miR-17-3p, miR-23b, miR-27a, miR-28, miR-29b, miR-30b, miR-31, miR-34b,miR-34c, miR-95, miR-96, miR-100, miR-104, miR-105, miR-106a, miR-107,miR-122a, miR-124a, miR-125b, miR-127, miR-128a, miR-128b, miR-129,miR-135a, miR-135b, miR-137, miR-138, miR-139, miR-140, miR-141,miR-149, miR-154, miR- 154#3, miR-181a, miR-182, miR-183, miR-184,miR-185, miR-189, miR-190, miR-199, miR-199b, miR-200a, miR-200b,miR-204, miR-213, miR-215, miR-216, miR-219, miR- 222, miR-224, miR-299,miR-302a, miR-302b, miR-302c, miR-302d, miR-323, miR-325, let-7a,let-7b, let-7d, let-7e, or let-7g; MUM-1, beta-catenin, or Nop/5/Sik;DUSP-1, Alix, hsp70, Gib2, Gia, moesin, GAPDH, malate dehydrogenase,pl20 catenin, PGRL, syntaxin- binding protein 1 & 2, septin-2, orWD-repeat containing protein 1; H/ACA (U1071), SNORA11D Head and neckcancer: miR-21, let-7, miR-18, miR-29c, miR-142-3p, miR-155, miR- 146b,miR-205, or miR-21; miR-494; HPV E6, HPV E7, p53, IL-8, SAT, H3FA3;EGFR, EphB4, orEphB2; CHCHD7-PLAG1, CTNNB1-PLAG1, FHIT-HMGA2,HMGA2-NFIB, LIFR-PLAG1, or TCEA1-PLAG1 Oral squamous cell carcinoma: p53autoantibodies, defensing-1, lncRNAs (MEG-3, MALAT-1, HOTAIR, NEAT-1,UCA) Cortisol, lactate dehydrogenase, Transferrin, cyclin Dl, Maspin,alpha-amylase, IL-8, TNF-α, IL-1, IL-6, Basic fibroblast growth factor,Statherin, Cyfra 21.1, TP A, CA125, Endothelin-1, IL-1β, CD44, IGF-1,MMP-2, MMP-9, CD59, Catalase, Profilin, S100A9/MRP14, M2BP, CEA,Carcinoma associated antigen CA-50, Salivary carbonyls, Maspin,8-oxoguanine DNA glycosylase, OGGI, Phosphorylated-Src, Ki-67, Zincfinger protein 501 peptide, Hemopexin, Haptoglobin, Complement C3,Transthyretin, al-antitrypsin, Peroxidase, GST, SOD, 8-OHdG,Glutathione, MD A, miR-125a, miR-200a, miR-31 Salivary gland tumors:Fibroblast growth factor 2 (FGF2) and fibroblast growth factor receptor1 (FGFRI) Hepatocellular carcinoma: miR-221; et-7a-1, let-7a-2,let-7a-3, let-7b, let-7c, let-7d, let- 76, let-7f-2, let-fg, miR-122a,miR-124a-2, miR-130a, miR-132, miR-136, miR-141, miR- 142, miR-143,miR-145, miR-146, miR-150, miR-155(BIC), miR-181a-l, miR-181a-2,miR-181c, miR-195, miR-199a-l-5p, miR-199a-2-5p, miR-199b, miR-200b,miR-214, miR-223, or pre-miR-594; miR-122, miR-100, or miR-10a; miR-198or miR-145 Renal cell carcinoma: miR-141, miR-200; miR-28, miR-185,miR-27, miR-let-7f-2; laminin receptor 1, betaig-h3, Galectin-1, a-2Macroglobulin, Adipophilin, Angiopoietin 2, Caldesmon 1, Class 11MHC-associated invariant chain (CD74), Collagen IV-al, Complementcomponent, Complement component 3, Cytochrome P450, subfamily IIJpolypeptide 2, Delta sleep-inducing peptide, Fc g receptor 111a (CD 16),HLA-B, HLA- DRa, HLA-DRb, HLA-SB, IFN-induced transmembrane protein 3,IFN-induced transmembrane protein 1, or Lysyl Oxidase; IF1 alpha, VEGF,PDGFRA; ALPHA-TFEB, NONO-TFE3, PRCC-TFE3, SFPQ-TFE3, CLTC-TFE3, orMALAT1-TFEBf Renal cell carcinoma: Akt, total Erk1/2, total Met, totalGSK3b, total Hifla, total p21, total AMPKa1, total VEGF, total P1GF,total VEGFR-1/Flt-1, phosphorylated Akt, phosphorylated Erk1/2,phosphorylated. Met, phosphorylated STAT3, phosphorylated GSK3b, andphosphorylated AMPKa1 Cervical cancer: HPV E6, HPV E7, or p53 Thyroidcancer: AKAP-BRAF, CCDC6-RET, ERC1-RETM, GOLGA5-RET, HOOK3- RET,HRH4-RET, KTN1-RET, NCOA4-RET, PCM1-RET, PRKARA1A-RET, RFG- RET,RFG9-RET, Ria-RET, TGF-NTRK1, TPM3-NTRK1, TPM3-TPR, TPR-MET, TPR- NTRK1,TRIM24-RET, TRIM27-RET or TRIM33-RET; PAX8-PPARy Neuroblastoma:Neuron-specific enolase (NSE) Glioblastoma: GFAP Brain cancer: miR-21,miR-10b, miR-130a, miR-221, miR-125b-1, miR-125b-2, miR-9- 2, miR-21,miR-25, or miR-123; miR-128a, miR-181c, miR-181a, or miR-181b; GOPC-ROS1; MGMT; EGFR Blood Cancers: HOX11, TALI, LY1, LMO1, or LMO2;TTL-ETV6, CDK6-MLL, CDK6-TLX3, ETV6-FLT3, ETV6-RUNX1, ETV6-TTL,MLL-AFF1, MLL-AFF3, MLL- AFF4, MLL-GAS7, TCBA1-ETV6, TCF3-PBX1 orTCF3-TFPT, for acute lymphocytic leukemia (ALL); BCL11B-TLX3,IL2-TNFRFS17, NUP214-ABL1, NUP98-CCDC28A, TAL1-STIL, or ETV6-ABL2, forT-cell acute lymphocytic leukemia (T-ALL); ATIC- ALK, KIAA1618-ALK,MSN-ALK, MYH9-ALK, NPM1-ALK, TGF-ALK or TPM3- ALK, for anaplastic largecell lymphoma (ALCL); BCR-ABL1, BCR-JAK2, ETV6-EVI1, ETV6-MN1 orETV6-TCBA1, for chronic myelogenous leukemia (CML); CBFB-MYH11,CHIC2-ETV6, ETV6-ABL1, ETV6-ABL2, ETV6-ARNT, ETV6-CDX2, ETV6-HLXB9,ETV6-PER1, MEF2D-DAZAP1, AML-AFF1, MLL-ARHGAP26, MLL-ARHGEF12,MLL-CASC5, MLL-CBL, MLL-CREBBP, MLL-DAB21P, MLL-ELL, MLL-EP300,MLL-EPS15, MLL-FNBPl, MLL-F0X03A, MLL-GMPS, MLL-GPHN, MLL-MLLT1,MLL-MLLT11, MLL-MLLT3, MLL-MLLT6, MLL-MYOIF, MLL-PICALM, MLL- SEPT2,MLL-SEPT6, MLL-SORBS2, MYST3-SORBS2, MYST-CREBBP, NPMI- MLF1,NUP98-HOXA13, PRDM16-EVI1, RABEP1-PDGFRB, RUNXI-EVI1, RUNX1- MDS1,RUNX1-RPL22, RUNX1-RUNX1T1, RUNX1-SH3D19, RUNX1-USP42, RUNX1-YTHDF2,RUNX1-ZNF687, or TAF15-ZNF-384, for AML; CCND1-FSTL3, for chroniclymphocytic leukemia (CLL); and FLIP1-PDGFRA, FLT3-ETV6, KIAA1509-PDGFRA, PDE4DIP-PDGFRB, NIN-PDGFRB, TP53BP1-PDGFRB, or TPM3-PDGFRB, forhyper eosinophilia/chronic eosinophilia; miR-23b, miR-24-1, miR-146,miR-155, miR- 195, miR-221, miR-331, miR-29a, miR-195, miR-34a, ormiR-29c; miR-15a, miR-16-1, miR-29 or miR-223; miR-128b, miR-204,miR-218, miR-331, miR-181b-l, miR-17-92 B-Cell Chronic LymphocyticLeukemia: miR-183-prec, miR-190, miR-24-1-prec, miR- 33, miR-19a,miR-140, miR-123, miR-10b, miR-15b-prec, miR-92-1, miR-188, miR-154,miR-217, miR-101, miR-141-prec, miR-153-prec, miR-196-2, miR-13 4,miR-141, miR- 132, miR-192, or miR-181b-prec; miR-213, miR-220; ZAP70,AdipoR1; BCL3-MYC, MYC-BTG1, BCL7A-MYC, BRWD3-ARHGAP20 orBTG1-MYC B-celllymphoma: miR-17-92 polycistron, miR-155, miR-210, or miR-21, miR-19a,miR- 92, miR-142 miR-155, miR-221 miR-17-92, miR-21, miR-191, miR-205,U50; miR-17-92, miR-155, miR-210, or miR-21; A-myb, LM02, JNK3, CD10,bcl-6, Cyclin D2, IRF4, Flip, or CD44; CITTA-BCL6, CLTC-ALK, IL21R-BCL6,PIM1-BCL6, TFCR-BCL6, IKZF1- BCL6 or SEC31A-ALK Burkitt’s lymphoma:pri-miR-155; MYC, TERT, NS, NP, MAZ, RCF3, BYSL, IDE3, CDC7, TCL1A,AUTS2, MYBL1, BMP7, ITPR3, CDC2, BACK2, TTK, MME, ALOX5, or TOP1; BCL6,KI-67; IGH-MYC, LCP1-BCL6 Endometrial cancer: miR-185, miR-106a,miR-181a, miR-210, miR-423, miR-103, miR- 107, or let-7c; miR-71,miR-221, miR-193, miR-152, or miR-30c; NLRP7, AlphaVBeta6 integrinUterine leiomyomas: let-7 family member, miR-21, miR-23b, miR-29b, ormiR-197 Myelofibrosis: miR-190; miR-31, miR-150 and miR-95; miR-34a,miR-342, miR-326, miR-105, miR-149, miR-147 Pheochromocytoma:Catecholamines (epinephrine, norepinephrine, adrenaline) Kidneydisease/injury: ADBP-26, NHE3, KIM-1, glutamyltransferase,N-acetyl-beta-D- glucosaminidase, lysozyme, NGAL, L-FABP, bikunin, urea,prostaglandins, creatinine, alpha-1-microglobulin, retinol bindingprotein, glutathione-S-transferases, adiponectin, beta-2-macroglobuin,calbindin-D, cysteine-rich angiogenic inducer 61, endothelial/epithialgrowth factors, alpha-1-acid glycoprotein (orosomucoid), prealbumin,modified albumin, albumin, transferrin, alpha-1-lipoprotein,alpha-1-antitrypsin matrix metalloproteinases (MMPs),alpha-1-fetoprotein, Tamm Horsfall protein, homoarginine, interleukin18, monocyte chemotactic protein-1 (MCP-1), Lipocalin, VCAN, NRP1, CCL2,CCL19, COL3A1, GZMM, alpha-galactosidase, casein kinase 2, IP-10, Mig,I- TAC, MIP-1α, MIP-3α, and MIP-1β, alpha-2-glycoprotein-Zinc,leucine-rich alpha-2- glycoprotein, uromodulin, Pacsin 2, hepcidin-20,hepcidin-25, AIF-2, urinary type-IV collagen, lipocalin-typeprostaglandin D synthase (L-PGDS), urinary neutrophil gelatinase-associated lipocalin (uNGAL), Annexin A1, Rab23, Shh, Ihh, Dhh, PTCH1,PTCH2, SMO, Gli1, Gli2, Gli3, TLR4, cystatin C, AQPl, AQP2, AQP3, NKCC2,NaPill, DAHKSEVAHRFKD; [RNA:] SLC12A1, UMOD, vWF, MMPl, MMP3, SLC22A6,SLC22A 8, SLC22A 12, podocin, cubulin, LRP2, AQP9, and albumin,carcinoembryonic antigen (CEA), mucin, alpha-fetoprotein, tyrosinase,melanoma associated antigen, mutated tumor protein 53, p21, PUMA,prostate-specific antigen (PSA) or thyroglobulin, von Willebrand factor(VWF), thrombin, factor VIII, plasmin, fibrin, osteopontin (SPP1),Rab23, Shh, Ihh, Dhh, PTCH1, PTCH2, SMO, Gli1, Gli2, Gli3 Liverfailure/disease: Lactoferrin, uric acid, cortisol, alpha-amylase,Carnitine; Cholic Acid; Chenodeoxycholic, Deoxycholic, Lithocholic,Glycocholic; Prostaglandin E₂; 13,14- dihydro-15-keto Prostaglandin A2;Prostaglandin B2; Prostaglandin F2a; 15-keto- Prostaglandin F2α;6-keto-Prostaglandin F1α; Thromboxane B2; 11-dehydro- Thromboxane B2;Prostaglandin D2; Prostaglandin J2; 15-deoxy-A12,14-Prostaglandin J2;11β-Prostaglandin F2α; 5(S)-Hydroxyeicosatetraenoic acid;5(S)-Hydroxyeicosapentaenoic acid; Leukotriene B4; Leukotriene B5;Leukotriene C4; Leukotriene D4; Leukotriene E4; Leukotriene F4;12(S)-Hydroxyeicosatetraenoic acid; 12(S)-Hydroxyeicosapentaenoic acid;15(S)-Hydroxyeicosatetraenoic acid; 15(S)-Hydroxy eicosapentaenoic acid;Lipoxin A4; 8(S)-Hydroxy eicosatetraenoic acid;9-Hydroxyeicosatetraenoic acid; 11- Hydroxyeicosatetraenoic acid;8-iso-Prostaglandin F2α; 9-Hydroxyoctadecadienoic acid; 13-Hydroxyoctadecadienoic acid; 20(S)-Hydroxyeicosatetraenoic acid; 9,10-Epoxyoctadecenoic acid; 12,13-Epoxyoctadecenoic acid;12,13-Dihydroxyoctadecenoic acid; 5,6-Epoxyeicosatrienoic acid;11,12-Epoxyeicosatrienoic acid; 14,15- Epoxyeicosatrienoic acid;5,6-Dihydroxyeicosatrienoic acid; 8,9-Dihydroxyeicosatrienoic acid;11,12-Dihydroxyeicosatrienoic acid; 14,15-Dihydroxyeicosatrienoic acid;14,15- Epoxyeicosatetraenoic acid; 17,18-Epoxyeicosatetraenoic acid;14,15- Dihydroxy eicosatetraenoic acid; 17,18-Dihydroxy eicosatetraenoicacid; 19,20- Dihydroxydocosapentaenoic acid; diacetylspermine,hemopexin, TLR4 Stroke: MMP9, S100-P, S100A12, SI00A9, coag factor V,Arginasel, CA-IV, monocarboxylic acid transporter, ets-2, EIF2alpha,cytoskeleton associated protein 4, N- formylpeptide receptor,Ribonuclease2, N-acetylneuraminate pyruvate lyase, BCL-6, or Glycogenphosphorylase Heart failure/Cardiovascular health: 8-iso-prostaglandinF2α (8-iso-PGF2a), miR-195, miR-208, miR-214, let-7b, let-7c, let-7e,miR-15b, miR-23a, miR-24, miR-27a, miR-27b, miR-93, miR-99b, miR-100,miR-103, miR-125b, miR-140, miR-145, miR-181a, miR- 191, miR-195,miR-199a, miR-320, miR-342, miR-451, or miR-499; miR-1, miR-10a,miR-17-5p, miR-19a, miR-19b, miR-20a, miR-20b, miR-26b, miR-28,miR-30e-5p, miR- 101, miR-106a, miR-126, miR-222, miR-374, miR-422b, ormiR-423; MRP 14, CD69; CK- MB, cTnI (cardiac troponin), CRP, BPN, IL-6,MCSF, CD40, CD40L, SFRP-3, NT- proBNP, troponin T, SKITHRIHWESASLL,AHKSEVAHRFK, uroguanylin, BNP, miR- 378, miR-497, miR-21, miR-99a, miR29a, miR-30b, miR-29c, miR-331.3p, miR-19a, miR-22, miR-502.3, andmiR-652; IL-16, sFas, Fas ligand, MCP-3, HGF, CTACK, EOTAXIN,adiponectin, IL-18, TIMP.4, TIMP.1, CRP, VEGF, and EGF, C-reactiveprotein (CRP); myoglobin (MYO), creatinine kinase myocardial band(CK-MB), cardiac troponins (cTn), and myeloperoxidase; TNF-a, and MMP-9;CD40 Vulnerable plaque: Amylase, L-6, MMP-9, PAPP-A, D-dimer,fibrinogen, Lp-PLA2, SCD40L, Il-18, oxLDL, GPx-1, MCP-1, P1GF, or CRPHigh blood pressure: lysozyme Fibromyalgia: NR2D Neuropathic Pain:CCR2/4, CNP; ICAM-1, CGRP, TIMP-1, CLR-1, HSP-27, FABP, orapolipoprotein D; OX42, ED9 Tiredness/fatigue:PPGKPQGPPPQGGNQPQGPPPPPGKPQ (SEQ ID NO: //); GNPQGPSPQGGNKPQGPPPPPGKPQ(SEQ ID NO: //); SPPGKPQGPPQQEGNKPQGPPPPGKPQ (SEQ ID NO: //); GGHPPPP(SEQ ID NO: //), ESPSLIA (SEQ ID NO: //); endorepellin; humanherpesvirus 6, human herpesvirus 7, human cytomegalovirus, andEpstein-Barr virus (EBV) Stress: Cortisol, chromogranin A,alpha-amylase, secretary IgA, lysozyme, dehydro- androsteronesulfate;17-ketosteroidsulfate; dehydro-epiandrostronesulfate; corticosteroid,17-hydroxycorticosteroid, growth hormone, oxytocin, aldose reductase,apoptosis signal- regulating kinase 1, aquaporin 5, beta-endorphin,betaine GABA transporter, caspase recruitment domain protein 9, caspase8, cyclin D, cyclooxygenase 2, cytochrome P450, cytochrome c, c-fos,c-jun, epidermal growth factor receptor, ferritin, glucocorticoidreceptor, glucose regulated protein 58, glucose regulated protein 75,glutathione S- transferase p, GroEL, heat shock protein 25/27, heatshock protein 40, heat shock protein 60, heat shock protein 70, heatshock protein 90, heat shock transcription factor-1, heme oxygenase-1,interleukin 1β, interleukin 6, interleukin 8, interleukin 10,interleukin 12, laminin, leptin receptor, matrix metalloproteinase 9,metallothionein, Mek-1, Mekk-1, inducible nitric oxide synthase,peripheral benzodiazepine receptor, p38 MAPK, salivary alpha amylase,SAPK, serotonin, serotonin receptor, substance P, superoxide dismutaseMn, superoxide dismutase Cu/Zn, superoxide dismutase EC, transforminggrowth factor β, tumor suppressor p53, and vasoactive intestinal peptideMalnutrition: sIgA Nutritional status: Prealbumin, Albumin,Retinol-binding protein (RBP), Transferrin, Acylation-StimulatingProtein (ASP), Adiponectin, Agouti-Related Protein (AgRP), Angiopoietin-like Protein 4 (ANGPTL4, FIAF), C-peptide, AFABP (Adipocyte FattyAcid Binding Protein, FABP4), Acylation-Stimulating Protein (ASP), EFABP(Epidermal Fatty Acid Binding Protein, FABP5), Glicentin, Glucagon,Glucagon-Like Peptide-1, Glucagon- Like Peptide-2, Ghrelin, Insulin,Leptin, Leptin Receptor, PYY, RELMs, Resistin, and sTfR (solubleTransferrin Receptor) Energy balance (protein excretion) / energy status/ metabolic state: AMPK, pre- albumin, retinol binding protein, urea,cholesterol, lipoproteins, insulin, insulin C peptide, IGF bindingproteins, e.g. IGF-BPl, liver enzymes Diabetes: 11-8, CTSS, ITGB2,HLA-DRA, CD53, PLAG27, or MMP9; RBP4; 8-iso- prostaglandin F2α(8-iso-PGF2α), 11-dehydro-thromboxane B₂ (TXM), C-peptide, Advancedglycosylation end products (AGEs), 1,5-anhydroglucitol, NGPTL3 and 4,autoantibodies (Zn transporter 8, glutamic acid decarboxylase (GAD)),ATP-binding cassette, sub-family C (CFTR/MRP), member 8; ATP-bindingcassette, sub-family C (CFTR/MRP), member 9; angiotensin I convertingenzyme (peptidyl-dipeptidase A) 1; adenylate cyclase activatingpolypeptide 1 (pituitary); adiponectin, C1Q and collagen domaincontaining; adiponectin receptor 1; adiponectin receptor 2;adrenomedullin; adrenergic, beta-2-, receptor, surface; advancedglycosylation end product-specific receptor; agouti related proteinhomolog (mouse); angiotensinogen (serpin peptidase inhibitor, clade A,member 8); angiotensin II receptor, type 1; angiotensin II receptor-associated protein; alpha-2-HS-glycoprotein; v-akt murine thymoma viraloncogene homolog 1; v-akt murine thymoma viral oncogene homolog 2;albumin; Alstrom syndrome 1; archidonate 12-lipoxygenase; ankyrin repeatdomain 23; apelin, AGTRL 1 Ligand; apolipoprotein A-I; apolipoproteinA-II; apolipoprotein B (including Ag(x) antigen); apolipoprotein E; arylhydrocarbon receptor nuclear translocator; Aryl hydrocarbon receptornuclear translocator-like; arrestin, beta 1; arginine vasopressin(neurophysin II, antidiuretic hormone, Diabetes insipidus,neurohypophyseal); bombesin receptor subtype 3; betacellulin;benzodiazepine receptor (peripheral); complement component 3; complementcomponent 4A (Rodgers blood group); complement component 4B (Childoblood group); complement component 5; Calpain-10; cholecystokinin;cholecystokinin (CCK)-A receptor; chemokine (C-C motif) ligand 2; CD14molecule; CD163 molecule; CD36 molecule (thrombospondin receptor); CD38molecule; CD3d molecule, delta (CD3- TCR complex); CD3g molecule, gamma(CD3-TCR complex); CD40 molecule, TNF receptor superfamily member 5;CD40 ligand (TNF superfamily, member 5, hyper-IgM syndrome); CD68molecule; cyclin-dependent kinase 5; complement factor D (adipsin);CASP8 and FADD-like apoptosis regulator; Clock homolog (mouse); chymase1, mast cell; cannabinoid receptor 1 (brain); cannabinoid receptor 2(macrophage); cortistatin; carnitine palmitoyltransferase 1; carnitinepalmitoyltransferase II; complement component (3b/4b) receptor 1;complement component (3d/Epstein Barr virus) receptor 2; CREB bindingprotein (Rubinstein-Taybi syndrome); C-reactive protein,pentraxin-related; CREB regulated transcription coactivator 2; colonystimulating factor 1 (macrophage); cathepsin B; cathepsin L; cytochromeP450, family 19, subfamily A, polypeptide 1; Dio-2, deathinducer-obliterator 1; dipeptidyl-peptidase 4 (CD26, adenosine deaminasecomplexing protein 2); epidermal growth factor (beta-urogastrone); earlygrowth response 1; epididymal sperm binding protein 1; ectonucleotide;pyrophosphatase/phosphodiesterase 1; E1A binding protein p300;coagulation factor XIII, A1 polypeptide; coagulation factor VIII,procoagulant component (hemophilia A); fatty acid binding protein 4,adipocyte; Fas (TNF receptor superfamily, member 6); Fas ligand (TNFsuperfamily, member 6); free fatty acid receptor 1; fibrinogen alphachain; forkhead box A2; forkhead box O1A; ferritin; glutamatedecarboxylase 2; galanin; gastrin; glucagon; glucokinase; gamma-glutamyltransferase 1; growth hormone 1; ghrelin/obestatinpreprohormone; gastric inhibitory polypeptide; gastric inhibitorypolypeptide receptor; glucagon-like peptide 1 receptor; guaninenucleotide binding protein (G protein), beta polypeptide 3; glutamic-pyruvate transaminase (alanine aminotransferase); gastrin releasingpeptide (bombesin); gelsolin (amyloidosis, Finnish type); hemoglobin;hemoglobin, beta; hypocretin (orexin); neuropeptide; precursor;hepatocyte growth factor (hepapoietin A; scatter factor); hepatocytenuclear factor 4, alpha; haptoglobin; hydroxy steroid (11-beta);dehydrogenase 1; heat shock 70 kDa protein 1B; islet amyloidpolypeptide; intercellular adhesion molecule 1 (CD54), human rhinovirusreceptor; interferon, gamma; insulin-like growth factor 1 (somatomedinC); insulin-like growth factor 2 (somatomedin A); insulin-like growthfactor binding protein 1; insulin-like growth factor binding protein 3;inhibitor of kappa light polypeptide gene enhancer in B-cells, kinasebeta; interleukin 10; interleukin 18 (interferon-gamma-inducing factor);interleukin 1, alpha; interleukin 1, beta; interleukin 1 receptorantagonist; interleukin 2; interleukin 6 (interferon, beta 2);interleukin 6 receptor; interleukin 8; inhibin, beta A (activin A,activin AB alpha polypeptide); insulin; insulin receptor; insulinpromoter factor-1; insulin receptor substrate 1; insulin receptorsubstrate-2; potassium inwardly-rectifying channel, subfamily J, member11; potassium inwardly-rectifying channel, subfamily J, member 8;klotho; kallikrein B, plasma (Fletcher factor) 1; leptin (obesityhomolog, mouse); leptin receptor; legumain; lipoprotein, Lp(a);lipoprotein lipase; v-maf musculoaponeurotic brosarcoma oncogene homologA (avian); mitogen-activated protein kinase 8; interacting protein 1;mannose-binding lectin (protein C) 2, soluble (opsonic defect);melanocortin 4 receptor; melanin-concentrating hormone receptor 1;matrix metallopeptidase 12 (macrophage elastase); matrixmetallopeptidase 14 (membrane-inserted); matrix metallopeptidase 2(gelatinase A, 72 kDa gelatinase, 72 kDa type IV collagenase); matrixmetallopeptidase 9 (gelatinase B, 92 kDa gelatinase, 92 kDa type IVcollagenase); nuclear receptor co- repressor 1; neurogenicdifferentiation 1; nuclear factor of kappa light polypeptide geneenhancer in B-cells 1 (p105); nerve growth factor, beta polypeptide;non-insulin-dependent Diabetes Mellitus (common, type 2) 1;non-insulin-dependent Diabetes Mellitus (common, type 2) 2;Noninsulin-dependent Diabetes Mellitus 3; nischarin (imidazolinereceptor); NF- kappaB repressing factor; neuronatin; nitric oxidesynthase 2A; Niemann-Pick disease, type C2; natriuretic peptideprecursor B; nuclear receptor subfamily 1, group D, member 1; nuclearrespiratory factor 1; oxytocin, prepro-(neurophysin 1); purinergicreceptor P2Y, G-protein coupled, 10; purinergic receptor P2Y, G-proteincoupled, 12; purinergic receptor P2Y, G-protein coupled, 2;progestagen-associated endometrial; protein (placental protein 14,pregnancy-associated endometrial alpha-2-globulin, alpha uterineprotein); paired box gene 4; pre-B-cell colony enhancing factor 1;phosphoenolpyruvate carboxykinase 1 (PEPCK1); proprotein convertase;subtilisin/kexin type 1; placental growth factor, vascular; endothelialgrowth factor-related protein; phosphoinositide-3-kinase, catalytic,alpha polypeptide; phosphoinositide-3-kinase, regulatory subunit 1 (p85alpha); phospholipase A2, group XIIA; phospholipase A2, group IID;plasminogen activator, tissue; patatin-like phospholipase domaincontaining 2; proopiomelanocortin(adrenocorticotropin/beta-lipotropin/alpha-melanocyte stimulatinghormone/beta- melanocyte stimulating hormone/beta-endorphin);paraoxonase 1 ESA, PON, Paraoxonase; peroxisome proliferative activatedreceptor, alpha; peroxisome proliferative activated receptor, delta;peroxisome proliferative activated receptor, gamma; peroxisomeproliferative activated receptor, gamma, coactivator 1; proteinphosphatase 1, regulatory (inhibitor) subunit 3 A (glycogen andsarcoplasmic reticulum binding subunit, skeletal muscle); proteinphosphatase 2A, regulatory subunit B' (PR 53); protein kinase, AMP-activated, beta 1 non-catalytic subunit; protein kinase, cAMP-dependent,catalytic, alpha; protein kinase C, epsilon; proteasome (prosome,macropain) 26S subunit, non-ATPase, 9 (Bridge-1); prostaglandin Esynthase; prostaglandin-endoperoxide synthase 2 (prostaglandin G/Hsynthase and cyclooxygenase); protein tyrosine phosphatase,mitochondrial 1; Peptide YY retinol binding protein 4, plasma (RBP4);regenerating islet- derived l alpha (pancreatic stone protein,pancreatic thread protein); resistin; ribosomal protein S6 kinase, 90kDa, polypeptide 1; Ras-related associated with Diabetes; serum amyloidAl; selectin E (endothelial adhesion molecule 1); serpin peptidaseinhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 6;serpin peptidase inhibitor, clade E (nexin, plasminogen activatorinhibitor type 1), member 1; serum/glucocorticoid regulated kinase; sexhormone-binding globulin; thioredoxin interacting protein; solutecarrier family 2, member 10; solute carrier family 2, member 2; solutecarrier family 2, member 4; solute carrier family 7 (cationic amino acidtransporter, y+ system), member 1(ERR); SNF1-like kinase 2; suppressorof cytokine signaling 3; v-src sarcoma (Schmidt-Ruppin A- 2) viraloncogene homolog (avian); sterol regulatory element bindingtranscription factor 1; solute carrier family 2, member 4; somatostatinreceptor 2; somatostatin receptor 5; transcription factor 1, hepatic;LF-B1, hepatic nuclear factor (HNF1); transcription factor 2, hepatic,LF-B3, variant hepatic nuclear factor; transcription factor 7-like 2(T-cell specific, HMG-box); transforming growth factor, beta 1(Camurati-Engelmann disease); transglutaminase 2 (C polypeptide,protein-glutamine-gamma-glutamyltransferase); thrombospondin 1;thrombospondin, type 1, domain containing 1; tumor necrosis factor (TNFsuperfamily, member 2); tumor necrosis factor (TNF superfamily, member2); tumor necrosis factor receptor superfamily, member 1A; tumornecrosis factor receptor superfamily, member IB; tryptophan hydroxylase2; thyrotropin-releasing hormone; transient receptor potential cationchannel, subfamily V, member 1; thioredoxin interacting protein;thioredoxin reductase 2; urocortin 3 (stresscopin); uncoupling protein 2(mitochondrial, proton carrier); upstream transcription factor 1;urotensin 2; vascular cell adhesion molecule 1; vascular endothelialgrowth factor; vimentin; vasoactive intestinal peptide; vasoactiveintestinal peptide receptor 1; vasoactive intestinal peptide receptor 2;von Willebrand factor; Wolfram syndrome 1 (wolframin); X-ray repaircomplementing defective repair in Chinese hamster cells 6; c-peptide;cortisol; vitamin D3; estrogen; estradiol; digitalis-like factor;oxyntomodulin; dehydroepiandrosterone sulfate (DHEAS); serotonin(5-hydroxytryptamine); anti-CD38 autoantibodies; gad65 autoantibody;Angiogenin, ribonuclease, RNase A family, 5; Hemoglobin A1c;Intercellular adhesion molecule 3 (CD50); interleukin 6 signaltransducer (gp130, oncostatin M receptor); selectin P (granule embraneprotein 140 kDa, antigen CD62); TIMP metallopeptidase inhibitor;Proinsulin; endoglin; interleukin 2 receptor, beta; insulin-like growthfactor binding protein 2; insulin-like growth factor 1 receptor;fructosamine, N-acetyl-beta-d-glucosaminidase, pentosidine, advancedglycation end product, beta2-microglobulin, pyrraline Metabolicsyndrome/prediabetes: GFAP autoantibodies Alcohol abuse/dependence:aminotransferases, gamma-glutamyltransferase, ethanol, ethylglucuronide, sialic acid, β-hexosaminidase A, oral peroxidase, methanol,diethyl ene/ethylene glycol, α-amylase, clusterin, haptoglobin,heavy/light chains of immunoglobulins and transferrin; α-fucosidase(FUC), α-mannosidase (MAN), β- galactosidase (GAL), and β-glucuronidase(GLU) Non-alcoholic fatty liver disease: cytokeratin CK-18 (M65antigen), caspase-cleaved CK- 18 (M30-antigen), resistin, adiponectin,visfatin, insulin, tumor necrosis factor-alpha (TNF- α), interleukin 6(IL-6), or interleukin 8 (IL-8), aspartate aminotransferase (AST) andalanine aminotransferase (ALT); gamma-glutamyltransferase (GGT),immunoglobulin A, carbohydrate-deficient transferrin (CDT), glutamicoxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT),bilirubin Cystic fibrosis: amylase, cathepsin-D, lactate dehydrogenaseEctodermal dysplasia: alpha-amylase Sarcoidosis: IL-6, TNF-α, IFN-α,IL-17, IP-10, MIG, HGF, VEGF, TNF-RII, G-CSF, IFN-γ, MCP-1, RANTES andIL-5 Asthma: eotaxin-1 /CCL11, RANTES/CCL5, and IL-5; IL-lp, IL-6,MCP-1/CCL2, and IL-8/CXCL8; IP-10/CXCL10 Periodontitis/dental caries:aspartate aminotransferase (AST) and alkaline phosphatase (ALP), uricacid and albumin; 12-HETE; MMP-8, TIMP-1, and ICTP Muscle damage:Myoglobin, creatine kinase (CK), lactate dehydrogenase (LDH), aldolase,troponin, carbonic anhydrase type 3 and fatty acid-binding protein(FABP), transaminases Infection (Mycobacterium tuberculosis): IL-32,NXNL1, PSMA7, C6orf61, EMP1, CLIC1, LACTB and DUSP3, LOC389541, MIDI IP1, KLRC3, KLF9, FBXQ32, C50RF29, CHUK , LOC652062, C6ORF60, MTMR11,sCD170; IFN-gamma; IL-Iβ, IL- 6, IL-8, IL-10, IL-12p70, sCD4, SCD25,SCD26, sCD32b/c, SCD50, SCD56, sCD66a, SCD83, sCD85j, SCD95, SCD106,sCD120b, sCD121b, SCD127, SCD154, SCD222, SCD226, sCDw329 and TNF alpha;VEGF, AAT, CRP, IL-IRA, TIMP-1, IL- 18, A2Macro, Haptoglobin ICAM-1,VCAM- 1, SCF, IL-17, Fibrinogen, beta-2- macroglobulin, TNF-alpha, C3and TNFR2, GPR117, TAZ, HSDL 1, HIP 1 (host) Infection (Helicobacterpylori): MUC-5B and MUC 7 Infection (Candida species): Hsp70,calprotectin, histatins, mucins, basic proline rich proteins andperoxidases (host); Infection (influenza): Hemagglutinin (H1),neuraminidase (N1); C-reactive protein, [RNA:] DNA cross-link repair 1A,PSO2 homolog, synaptonemal complex protein 3, v- maf musculoaponeuroticfibrosarcoma oncogene family, chitinase 3-like 3, matrixmetalloproteinase 12, ATP-binding cassette, sub-family E (OABP), member1, ATP- binding cassette, sub-family F (GCN20), member 1, feminization 1homolog a (C. elegans), general transcription factor IIH. polypeptide 2,forkhead box P1, zinc finger protein 282, arginyl-tRNA synthetase-like,Mitochondrial ribosomal protein L48, ribosomal protein S4, X-linked,eukaryotic translation elongation factor 1 alpha 1, proteaseome(prosome, macropain) 28 subunit 3, GLE1 RNA export mediator-like(yeast), small nuclear ribonucleoprotein polypeptide A′, cleavage andpolyadenylation specific factor 2, ribosomal protein L27a,, thioredoxindomain containing 4 (endoplasmic reticulum), flap structure specificendonuclease 1, ADP-ribosylation factor-like 6 interacting protein 2,cytidine 5′-triphosphate synthase 2, glutathione S-transferase, mu 5,phospholipase D1, aspartate-beta-hydroxylase, leukotriene A4 hydrolase,cytochrome P450 family 17, subfamily a, polypeptide 1, thioredoxininteracting protein, carbonyl reductase 2, alpha globin regulatoryelement containing gene, male-specific lethal-2 homolog (Drosophila),RAB1, member RAS oncogene family, protein tyrosine phosphatase, non-receptor type 21, potassium voltage-gated channel, lsk-relatedsubfamily, gene 3, Bcl2- associated athanogene 3, lymphocyte cytosolicprotein 2, pore forming protein-like, tumor necrosis factor receptorsuperfamily, member 19, filamin beta, microtubule-actin crosslinkingfactor 1, keratin complex 1, acidic, gene 18, keratin complex 1, acidic,gene 19, mesoderm development candiate 2, tubulin, alpha 4,, glutathioneperoxidase 1, integrin linked kinase, guanine nucleotide bindingprotein, alpha inhibiting 2, cyclin L2, tubulin, alpha 2, DEAD(Asp-Glu-Ala-Asp) box polypeptide 5, programmed cell death 4, proteasome(prosome, macropain) 26S subunit, non-ATPase 8, signal sequencereceptor, beta, RAD23b homolog (host) Infection (HIV-1): p24, gp41,gp120 Infection (Hepatitis B virus): Core, Envelope, Surface (Ay)Infection (Hepatitis C virus): Core, NS3, NS4, NS5 Infection (HepatitisE virus): orf2 3 KD, orf2 6 KD, orf3 3 KD Infection (Vibrio cholerae):Cholera Toxin Infection (Corynebacterium diphtheria): Diphtheria toxinInfection (Epstein-Barr virus): EA, VCA, NA Infection (Herpes simplexvirus HSV-1): gD Infection (Herpes simplex virus HSV-2): gG Infection(Clostridium tetani): Tetanus toxin Infection (Treponema pallidum): 15kd, p47 Infection (Entamoeba histolytica): M17 Infection (Toxoplasmagondii): a2-HS glycoprotein and apB glycoprotein (host); TGME49 052280,TGME49_021500, TGME49J) 19630, TGME49_061720 and TGME49_076220 Infection(Dengue virus): IL-10, fibrinogen, C4A, immunoglobulin, tropomyosin,albumin, SCSb-9 complement complex (host); NS-1 Infection (Streptococcuspneumonia): stratifin, cullin 1, selenoprotein K, metal response elementbinding transcription factor 2, prostaglandin E synthase 2, HLA-Bassociated transcript 4, zinc finger protein (C2H2 type) 276,GCIP-interacting protein p29, mitochondrial ribosomal protein L20, arylhydrocarbon receptor nuclear translocator-like, secretory carriermembrane protein 1, nuclear receptor subfamily 5, group A, member 2,NIMA (never in mitosis gene a)-related expressed, kinase 7, ribosomalprotein L28, ribosomal protein S25, lysosomal-associated proteintransmembrane 5, neural precursor cell expressed, developmentally,down-regulted gene 4, alpha glucosidase 2, alpha neutral subunit, coatomer protein complex, subunit beta 2 (beta prime), ribosomal protein L3,NADH dehydrogenase (ubiquinone) 1 alpha, subcomplex, assembly factor 1,isoprenylcysteine carboxyl methyltransferase, , cytoplasmicpolyadenylation element binding protein 3, mannosideacetylglucosaminyltransferase 1, RNA-binding region (RNP1, RRM)containing 1,, folate receptor 4 (delta), ATPase, H+ transporting,lysosomal 50/57 kDa, V1, subunit H, zinc finger, DHHC domain containing6, phosphoribosyl pyrophosphate synthetase-associated, protein 2,choline/ethanolaminephosphotransferase 1, solute carrier family 38,member 1, ATP synthase, H+ transporting, mitochondrial F0, complex,subunit f, isoform 2, glucose phosphate isomerase 1, 2′-5′oligoadenylate synthetase 1A, tyrosine hydroxylase, hemoglobin alpha,adult chain 1, selenoprotein P, plasma, 1, acetyl-Coenzyme Adehydrogenase, long-chain, mannosidase, beta A, lysosomal, deltex 3homolog (Drosophila), ras homolog gene family, member AB, estrogenreceptor 1 (alpha), phosphoglycerate kinase 1, keratin complex 2, basic,gene 8, emerin, nucleoporin 153, formin 2, prothymosin alpha, synapsinI, cullin 4B, regulator of chromosome condensation (RCC1) and, BTB (POZ)domain containing protein 1, immediate early response 5, SAM domain andHD domain, 1, tumor rejection antigen gp96, lymphocyte antigen 6complex, locus E, DAZ associated protein 2, general transcription factorII I, RNA polymerase II transcriptional coactivator, SWI/SNF-related,matrix-associated actin-dependent, regulator of chromatin, subfamily a,containing DEAD/H, box 1, structure specific recognition protein 1,ankyrin repeat and FYVE domain containing 1, SET translocation, myocyteenhancer factor 2A, homeo box D9, H2A histone family, member Z, cellularnucleic acid binding protein, golgi reassembly stacking protein 2,cathepsin L, eukaryotic translation initiation factor 5, ubiquitinspecific protease 9, X chromosome, proteasome (prosome, macropain)subunit, alpha type 7, pescadillo homolog 1, containing BRCT domain,(zebrafish), heterogeneous nuclear ribonucleoprotein K, DEAD(Asp-Glu-Ala-Asp) box polypeptide 52, sorting nexin 5, cathepsin B, DnaJ(Hsp40) homolog, subfamily B, member 9, ribosomal protein S3 a,,cytoplasmic polyadenylation element binding protein 4, 5′ -3′exoribonuclease 2, small nuclear ribonucleoprotein polypeptide F,,arachidonate 5-lipoxygenase activating protein, cytochrome c oxidase,subunit VIc, RIKubiquinol cytochrome c reductase core protein 2, lactatedehydrogenase 2, B chain, ubiquinol-cytochrome c reductase core protein1, ATP synthase, H+ transporting, mitochondrial F0, complex, subunit b,isoform 1, microsomal glutathione S-transferase 1, ras homolog genefamily, member A, RAB7, member RAS oncogene family, EGF-like modulecontaining, mucin-like, hormone, receptor-like sequence 1, annexin A6,mitogen activated protein kinase 3, tyrosine kinase, non-receptor, 2,villin 2, tubulin, beta 5, catenin src (host); Pneumolysin, pneumococcalhistidine triad D (PhtD), pneumococcal histidine triad E (PhtE), LytB,and pneumococcal choline-binding protein A (PepA) Infection (Mycoplasmapneumonia): DnaK, L7/L12, P1, exotoxin Infection (Campylobacter jejuni):gyrA, 16S rDNA, or flaA/flaB Infection (Bacillus anthracis): Lethalfactor, HtrA (BA3660), NlpC/P60-domain endopeptidase (BA1952), BA0796locus (BA0796), SAP Infection (West Nile virus): Infection (Humanpapilloma virus): E6, E7 Infection: RNase 7 (host)

In some embodiments, the devices, systems and methods of the inventioncan be used to inform a subject from whom a sample is derived about ahealth condition. Health conditions that may be diagnosed or measured bythe present methods, devices and systems include, but are not limitedto: chemical balance; nutritional health; exercise; fatigue; sleep;stress; prediabetes; allergies; aging; exposure to environmental toxins,pesticides, herbicides, synthetic hormone analogs; pregnancy; menopause;and andropause. Table 4.3 provides exemplary diagnostic markers that canbe detected using the present invention, and their associated healthconditions.

TABLE 4.3 Diagnostic markers Health Sample Condition Source Marker(s)Diabetes Saliva pIgR, Arp 3, CA VI, and IL-1Ra; PLS-2, LEI, and IGJchain, resistin Diabetes Miscellaneous ATP-binding cassette, sub-familyC (CFTR/MRP), member 8; ATP- binding cassette, sub-family C (CFTR/MRP),member 9; angiotensin I converting enzyme (peptidyl-dipeptidase A) 1;adenylate cyclase activating polypeptide 1 (pituitary); adiponectin, C1Qand collagen domain containing; adiponectin receptor 1; adiponectinreceptor 2; adrenomedullin; adrenergic, beta-2-, receptor, surface;advanced glycosylation end productspecific receptor; agouti relatedprotein homolog (mouse); angiotensinogen (serpin peptidase inhibitor,clade A, member 8); angiotensin II receptor, type 1; angiotensin IIreceptor-associated protein; alpha-2-HS-glycoprotein; v-akt murinethymoma viral oncogene homolog 1; v-akt murine thymoma viral oncogenehomolog 2; albumin; Alstrom syndrome 1; archidonate 12-lipoxygenase;ankyrin repeat domain 23; apelin, AGTRL 1 Ligand; apolipoprotein A-I;apolipoprotein A-II; apolipoprotein B (including Ag(x) antigen);apolipoprotein E; aryl hydrocarbon receptor nuclear translocator; Arylhydrocarbon receptor nuclear translocator-like; arrestin, beta 1;arginine vasopressin (neurophysin II, antidiuretic hormone, Diabetesinsipidus, neurohypophyseal); bombesin receptor subtype 3; betacellulin;benzodiazepine receptor (peripheral); complement component 3; complementcomponent 4A (Rodgers blood group); complement component 4B (Childoblood group); complement components; Calpain- 10; cholecystokinin;cholecystokinin (CCK)-A receptor; chemokine (C-C motif) ligand 2; CD14molecule; CD163 molecule; CD36 molecule (thrombospondin receptor); CD38molecule; CD3d molecule, delta (CD3-TCR complex); CD3g molecule, gamma(CD3-TCR complex); CD40 molecule, TNF receptor superfamily member 5;CD40 ligand (TNF superfamily, members, hyper- IgM syndrome); CD68molecule; cyclin-dependent kinase 5; complement factor D (adipsin);CASP8 and FADD-like apoptosis regulator; Clock homolog (mouse); chymase1, mast cell; cannabinoid receptor 1 (brain); cannabinoid receptor 2(macrophage); cortistatin; carnitine palmitoyltransferase I; carnitinepalmitoyltransferase II; complement component (3b/4b) receptor 1;complement component (3d/Epstein Barr virus) receptor 2; CREB bindingprotein (Rubinstein-Taybi syndrome); C-reactive protein,pentraxin-related; CREB regulated transcription coactivator 2; colonystimulating factor 1 (macrophage); cathepsin B; cathepsin L; cytochromeP450, family 19, subfamily A, polypeptide 1; Dio-2, deathinducer-obliterator 1; dipeptidyl-peptidase 4 (CD26, adenosine deaminasecomplexing protein 2); epidermal growth factor (beta-urogastrone); earlygrowth response 1; epididymal sperm binding protein 1; ectonucleotide;pyrophosphatase/phosphodiesterase 1; E1A binding protein p300;coagulation factor XIII, A1 polypeptide; coagulation factor VIII,procoagulant component (hemophilia A); fatty acid binding protein 4,adipocyte; Fas (TNF receptor superfamily, member 6); Fas ligand (TNFsuperfamily, member 6); free fatty acid receptor 1; fibrinogen alphachain; forkhead box A2; forkhead box O1A; ferritin; glutamatedecarboxylase 2; galanin; gastrin; glucagon; glucokinase;gamma-glutamyltransferase 1; growth hormone 1; ghrelin/obestatinpreprohormone; gastric inhibitory polypeptide; gastric inhibitorypolypeptide receptor; glucagonlike peptide 1 receptor; guaninenucleotide binding protein (G protein), beta polypeptide 3;glutamicpyruvate transaminase (alanine aminotransferase); gastrinreleasing peptide (bombesin); gelsolin (amyloidosis, Finnish type);hemoglobin; hemoglobin, beta; hypocretin (orexin); neuropeptide;precursor; hepatocyte growth factor (hepapoietin A; scatter factor);hepatocyte nuclear factor 4, alpha; haptoglobin; hydroxysteroid(11-beta); dehydrogenase 1; heat shock 70 kDa protein 1B; islet amyloidpolypeptide; intercellular adhesion molecule 1 (CD54), human rhinovirusreceptor; interferon, gamma; insulin-like growth factor 1 (somatomedinC); insulin-like growth factor 2 (somatomedin A); insulin- like growthfactor binding protein 1; insulin-like growth factor binding protein 3;inhibitor of kappa light polypeptide gene enhancer in B-cells, kinasebeta; interleukin 10; interleukin 18 (interferon-gamma inducing factor);interleukin 1, alpha; interleukin 1, beta; interleukin 1 receptorantagonist; interleukin 2; interleukin 6 (interferon, beta 2);interleukin 6 receptor; interleukin 8; inhibin, beta A (activin A,activin AB alpha polypeptide); insulin; insulin receptor; insulinpromoter factor-1; insulin receptor substrate 1; insulin receptorsubstrate-2; potassium inwardly-rectifying channel, subfamily J, member11; potassium inwardly-rectifying channel, subfamily J, member 8;klotho; kallikrein B, plasma (Fletcher factor) 1; leptin (obesityhomolog, mouse); leptin receptor; legumain; lipoprotein, Lp(a);lipoprotein lipase; v-maf musculoaponeurotic brosarcoma oncogene homologA (avian); mitogen-activated protein kinase 8; interacting protein 1;mannose-binding lectin (protein C) 2, soluble (opsonic defect);melanocortin 4 receptor; melanin-concentrating hormone receptor 1;matrix metallopeptidase 12 (macrophage elastase); matrixmetallopeptidase 14 (membrane-inserted); matrix metallopeptidase 2(gelatinase A, 72 kDa gelatinase, 72 kDa type IV collagenase); matrixmetallopeptidase 9 (gelatinase B, 92 kDa gelatinase, 92 kDa type IVcollagenase); nuclear receptor corepressor 1; neurogenic differentiation1; nuclear factor of kappa light polypeptide gene enhancer in B-cells1(p105); nerve growth factor, beta polypeptide; non-insulin-dependentDiabetes Mellitus (common, type 2) 1; non-insulin-dependent DiabetesMellitus (common, type 2) 2; Noninsulindependent Diabetes Mellitus 3;nischarin (imidazoline receptor); NF-kappaB repressing factor;neuronatin; nitric oxide synthase 2A; Niemann-Pick disease, type C2;natriuretic peptide precursor B; nuclear receptor subfamily 1, group D,member 1; nuclear respiratory factor 1; oxytocin, prepro-(neurophysinI); purinergic receptor P2Y, Gprotein coupled, 10; purinergic receptorP2Y, Gprotein coupled, 12; purinergic receptor P2Y, Gprotein coupled, 2;progestagen-associated endometrial; protein (placental protein 14,pregnancy-associated endometrial alpha-2-globulin, alpha uterineprotein); paired box gene 4; pre-B-cell colony enhancing factor 1;phosphoenolpyruvate carboxykinase 1 (PEPCK1); proprotein convertase;subtilisin/kexin type 1; placental growth factor, vascular; endothelialgrowth factor-related protein; phosphoinositide-3-kinase, catalytic,alpha polypeptide; phosphoinositide-3-kinase, regulatory subunit 1 (p85alpha); phospholipase A2, group XIIA; phospholipase A2, group IID;plasminogen activator, tissue; patatinlike phospholipase domaincontaining 2; proopiomelanocortin (adrenocorticotropin/betalipotropin/alpha-melanocyte stimulating hormone/beta-melanocyte stimulatinghormone/beta-endorphin); paraoxonase 1 ESA, PON, Paraoxonase; peroxisomeproliferative activated receptor, alpha; peroxisome proliferativeactivated receptor, delta; peroxisome proliferative activated receptor,gamma; peroxisome proliferative activated receptor, gamma, coactivator1; protein phosphatase 1, regulatory (inhibitor) subunit 3A (glycogenand sarcoplasmic reticulum binding subunit, skeletal muscle); proteinphosphatase 2A, regulatory subunit B□(PR 53); protein kinase,AMP-activated, beta 1 non-catalytic subunit; protein kinase,cAMP-dependent, catalytic, alpha; protein kinase C, epsilon; proteasome(prosome, macropain) 26S subunit, non-ATPase, 9 (Bridge-1);prostaglandin E synthase; prostaglandinendoperoxide synthase 2(prostaglandin G/H synthase and cyclooxygenase); protein tyrosinephosphatase, mitochondrial 1; Peptide YY retinol binding protein 4,plasma (RBP4); regenerating islet-derived 1 alpha (pancreatic stoneprotein, pancreatic thread protein); resistin; ribosomal protein S6kinase, 90 kDa, polypeptide 1; Rasrelated associated with Diabetes;serum amyloid A1; selectin E (endothelial adhesion molecule 1); serpinpeptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin),member 6; serpin peptidase inhibitor, clade E (nexin, plasminogenactivator inhibitor type 1), member 1; serum/glucocorticoid regulatedkinase; sex hormone-binding globulin; thioredoxin interacting protein;solute carrier family 2, member 10; solute carrier family 2, member 2;solute carrier family 2, member 4; solute carrier family 7 (cationicamino acid transporter, y+ system), member 1(ERR); SNF1-like kinase 2;suppressor of cytokine signaling 3; v-src sarcoma (Schmidt- Ruppin A-2)viral oncogene homolog (avian); sterol regulatory element bindingtranscription factor 1; solute carrier family 2, member 4; somatostatinreceptor 2; somatostatin receptor 5; transcription factor 1, hepatic;LF-B1, hepatic nuclear factor (HNF1); transcription factor 2, hepatic,LF-B3, variant hepatic nuclear factor; transcription factor 7-like 2(T-cell specific, HMGbox); transforming growth factor, beta 1 (Camurati-Engelmann disease); transglutaminase 2 (C polypeptide,protein-glutamine-gammaglutamyltransferase); thrombospondin 1;thrombospondin, type I, domain containing 1; tumor necrosis factor (TNFsuperfamily, member 2); tumor necrosis factor (TNF superfamily, member2); tumor necrosis factor receptor superfamily, member 1A; tumornecrosis factor receptor superfamily, member 1B; tryptophan hydroxylase2; thyrotropin-releasing hormone; transient receptor potential cationchannel, subfamily V, member 1; thioredoxin interacting protein;thioredoxin reductase 2; urocortin 3 (stresscopin); uncoupling protein 2(mitochondrial, proton carrier); upstream transcription factor 1;urotensin 2; vascular cell adhesion molecule 1; vascular endothelialgrowth factor; vimentin; vasoactive intestinal peptide; vasoactiveintestinal peptide receptor 1; vasoactive intestinal peptide receptor 2;von Willebrand factor; Wolfram syndrome 1 (wolframin); X-ray repaircomplementing defective repair in Chinese hamster cells 6; c-peptide;cortisol; vitamin D3; estrogen; estradiol; digitalis-like factor;oxyntomodulin; dehydroepiandrosterone sulfate (DHEAS); serotonin(5-hydroxytryptamine); anti-CD38 autoantibodies; gad65 autoantibody;Angiogenin, ribonuclease, RNase A family, 5; Hemoglobin A1c;Intercellular adhesion molecule 3 (CD50); interleukin 6 signaltransducer (gp130, oncostatin M receptor); selectin P (granule embraneprotein 140 kDa, antigen CD62); TIMP metallopeptidase inhibitor;Proinsulin; endoglin; interleukin 2 receptor, beta; insulin-like growthfactor binding protein 2; insulin-like growth factor 1 receptor;fructosamine, N-acetyl-beta-d-glucosaminidase, pentosidine, advancedglycation end product, beta2-microglobulin, pyrraline Metabolic SerumGFAP autoantibodies syndrome/ prediabetes Kidney Saliva Lactoferrin,uric acid, cortisol, alpha-amylase failure/disease Kidney MiscellaneousADBP-26, NHE3, KIM-1, glutamyltransferase, Nacetyl- failure/diseasebeta-D-glucosaminidase, lysozyme, NGAL, L-FABP, bikunin, urea,prostaglandins, creatinine, alpha-1-microglobulin, retinol bindingprotein, glutathione-S-transferases, adiponectin, beta-2- macroglobuin,calbindin-D, cysteine-rich angiogenic inducer 61, endothelial/epithialgrowth factors, alpha-1-acid glycoprotein (orosomucoid), prealbumin,modified albumin, albumin, transferrin, alpha-1-lipoprotein,alpha-1-antitrypsin matrix metalloproteinases (MMPs),alpha-1-fetoprotein, Tamm Horsfall protein, homoarginine, interleukin18, monocyte chemotactic protein-1 (MCP-1), Lipocalin, VCAN, NRP1, CCL2,CCL19, COL3A1, GZMM, alpha-galactosidase, casein kinase 2, IP-10, Mig,I-TAC, MIP-1α, MIP-3α, MIP-1β, alpha-2-glycoprotein-Zinc, leucine-richalpha-2- glycoprotein, uromodulin, Pacsin 2, hepcidin-20, hepcidin-25,AIF-2, urinary type-IV collagen, lipocalin-type prostaglandin D synthase(L-PGDS), urinary neutrophil gelatinase-associated lipocalin (uNGAL),Annexin A1, Rab23, Shh, Ihh, Dhh, PTCH1, PTCH2, SMO, Gli1, Gli2, Gli3,TLR4, cystatin C, AQPI, AQP2, AQP3, NKCC2, NaPill, DAHKSEVAHRFKD RNA:]SLC12A1, UMOD, vWF, MMPI, MMP3, SLC22A6, SLC22A 8, SLC22A 12, podocin,cubulin, LRP2, AQP9, and albumin, carcinoembryonic antigen (CEA), mucin,alphafetoprotein, tyrosinase, melanoma associated antigen, mutated tumorprotein 53, p21, PUMA, prostate-specific antigen (PSA) or thyroglobulin,von Willebrand factor (VWF), thrombin, factor VIII, plasmin, fibrin,osteopontin (SPP1), Rab23, Shh, Ihh, Dhh, PTCH1, PTCH2, SMO, Gli1, Gli2,Gli3 Liver Miscellaneous Carnitine; Cholic Acid; Chenodeoxycholic,failure/disease Deoxycholic, Lithocholic, Glycocholic; Prostaglandin E2;13,14-dihydro-15-keto Prostaglandin A2; Prostaglandin B2; ProstaglandinF2a; 15-keto- prostaglandin F2α; 6-keto-prostaglandin F1α, ThromboxaneB2; Prostaglandin D2; Prostaglandin J2; 15-deoxy-Δ12,14-prostaglandinJ2; 11β-prostaglandin F2α; 5(S)-Hydroxyeicosatetraenoic acid;5(S)-Hydroxyeicosapentaenoic acid; Leukotriene B4; Leukotriene B5;Leukotriene C4; Leukotriene D4; Leukotriene E4; Leukotriene F4;12(S)-Hydroxyeicosatetraenoic acid; 12(S)- Hydroxyeicosapentaenoic acid;15(S)- Hydroxyeicosatetraenoic acid; 15(S)- Hydroxyeicosapentaenoicacid; Lipoxin A4; 8(S)- Hydroxyeicosatetraenoic acid; 9-Hydroxyeicosatetraenoic acid; 11- Hydroxyeicosatetraenoic acid;8-iso-Prostaglandin F2α; 9-Hydroxyoctadecadienoic acid; 13-Hydroxyoctadecadienoic acid; 20(S)- Hydroxyeicosatetraenoic acid; 9,10-Epoxyoctadecenoic acid; 12,13-Epoxyoctadecenoic acid;12,13-Dihydroxyoctadecenoic acid; 5,6- Epoxyeicosatrienoic acid; 11,12-Epoxyeicosatrienoic acid; 14,15- Epoxyeicosatrienoic acid; 5,6-Dihydroxyeicosatrienoic acid; 8,9- Dihydroxyeicosatrienoic acid; 11,12-Dihydroxyeicosatrienoic acid; 14,15- Dihydroxyeicosatrienoic acid;14,15- Epoxyeicosatetraenoic acid; 17,18- Epoxyeicosatetraenoic acid;14,15- Dihydroxyeicosatetraenoic acid; 17,18- Dihydroxyeicosatetraenoicacid; 19,20- Dihydroxydocosapentaenoic acid; diacetylspermine,hemopexin, TLR4 Heart Miscellaneous SFRP-3, NT-proBNP, troponin T,Failure SKITHRIHWESASLL, AHKSEVAHRFK, uroguanylin, BNP CardiovascularMiscellaneous miR-378, miR-497, miR-21, miR-15b, miR-99a, health miR29a, miR-24, miR-30b, miR-29c, miR-331.3p, miR-19a, miR-22, miR-126,let-7b, miR-502.3, and miR-652, IL-16, sFas, Fas ligand, MCP-3, HGF,CTACK, EOTAXIN, adiponectin, IL-18, TIMP.4, TIMP.1, CRP, VEGF, and EGFCardiovascular Saliva C-reactive protein (CRP); myoglobin (MYO), healthcreatinine kinase myocardial band (CK-MB), cardiac troponins (cTn), andmyeloperoxidase; TNF-α, MMP-9; CD40 High Saliva lysozyme blood pressureTiredness/ Urine Endorepellin, human herpesvirus 6, human herpesvirus 7,human fatigue cytomegalovirus, and Epstein-Barr virus (EBV) Tiredness/Saliva PPGKPQGPPPQGGNQPQGPPPPPGKPQ; fatigue GNPQGPSPQGGNKPQGPPPPPGKPQ;SPPGKPQGPPQQEGNKPQGPPPPGKPQ Tiredness/ Miscellaneous GGHPPPP, ESPSLIAfatigue Malnutrition Saliva sIgA Depressive Miscellaneous Secretogranin,VGF disorder Alzheimer's CSF, β-amyloid(1-42), β-amyloid(1-40), tau,phosphortau- disease serum, 181 saliva Stress Saliva Cortisol,dehydro-androsteronesulfate; 17- ketosteroidsulfate;dehydro-epiandrostronesulfate; corticosteroid, 17-hydroxycorticosteroid,chromogranin A, alpha-amylase, secretary IgA, lysozyme, growth hormone,oxytocin Stress Miscellaneous aldose reductase, apoptosissignal-regulating kinase 1, aquaporin 5, beta-endorphin, betaine GABAtransporter, caspase recruitment domain protein 9, caspase 8, cyclin D,cyclooxygenase 2, cytochrome P450, cytochrome c, c-fos, c-jun, epidermalgrowth factor receptor, ferritin, glucocorticoid receptor, glucoseregulated protein 58, glucose regulated protein 75, glutathioneS-transferase p, GroEL, heat shock protein 25/27, heat shock protein 40,heat shock protein 60, heat shock protein 70, heat shock protein 90,heat shock transcription factor-1, heme oxygenase-1, interleukin 1β,interleukin 6, interleukin 8, interleukin 10, interleukin 12, laminin,leptin receptor, matrix metalloproteinase 9, metallothionein, Mek-1,Mekk-1, inducible nitric oxide synthase, peripheral benzodiazepinereceptor, p38 MAPK, salivary alpha amylase, SAPK, serotonin, serotoninreceptor, substance P, superoxide dismutase Mn, superoxide dismutaseCu/Zn, superoxide dismutase EC, transforming growth factor β, tumorsuppressor p53, vasoactive intestinal peptide Circadian Saliva melatoninrhythm Bone Urine Pyridinoline, deoxypyridinoline, collagen type 1turnover/ corss-linked N-telopeptide (NTX), collagen type 1 osteoporosiscorss-linked C-telopeptide (CTX), bone sialoprotein (BSP),Tartrate-resistant acid phosphatase 5b Bone Saliva deoxypyridinium(D-PYR) and osteocalcin (OC), turnover/ hepatocyte growth factor andinterleukin-1 beta osteoporosis Muscle Serum, Myoglobin, creatine kinase(CK), lactate damage urine dehydrogenase (LDH), aldolase, troponin,carbonic anhydrase type 3 and fatty acid-binding protein (FABP),transaminases Exercise/ Sweat Urea athletic activity Exercise/ SerumMyostatin, follistatin-like related gene athletic activity Exercise/Saliva Testosterone athletic activity Performance Miscellaneousinterleukin-6, interleukin-1 beta, G-CSF, interferongamma, enhancementinterleukin-8, interleukin-9, MCP-1, MIPbeta, and/or TNF alpha EnergySerum AMPK balance (protein excretion)/ Energy status/ metabolic stateEnergy Urine, pre-albumin, retinol binding protein, urea balance sweat,(protein feces excretion)/ Energy status/ metabolic state EnergyMiscellaneous cholesterol, lipoproteins, insulin, insulin C peptide,balance IGF binding proteins, e.g. IGF-BPI, liver enzymes (proteinexcretion)/ Energy status/ metabolic state Growth Saliva IGF-1Andropause Saliva testosterone; testosterone precursors suchaspregnenolone, progesterone, 17- hydroxypregnenolone,17-hydroxyprogesterone, dehydroepiandrosterone (DHEA) and delta-4-androstene-3,17-dione; testosterone and dihydrotestosterone metabolitessuch as the 17- ketosteroids androsterone and etiocholanolone, polarmetabolites in the form of diols, triols, and conjugates, as wellestradiol, estrogens, androsteindione, cortisol, DHEA, FSH (folliclestimulating hormone), LH (luteinizing hormone), and GnRH(gonadotropin-releasing hormone) Menopause Saliva Follicle stimulatinghormone (FSH) Estrogen and progesterone, testosterone, freetestosterone, and dehydroepiandrosterone sulfate (DHEAS), cortisol anddehydroepiandrosterone (DHEA) Pregnancy/ Saliva Progesterone fetaldevelopment Pregnancy/ Urine human chorionic gonadotropin,Levonorgestrel, fetal alpha-fetoprotein development Pregnancy/ SerumEstradiol fetal development Breast Urine 47D10 antigen, PTCD2, SLC25A20,NFKB2, cancer RASGRP2, PDE7A, MLL, PRKCE, GPATC3, PRIC285 and GSTA4,MIPEP, PLCB2, SLC25A19, DEF6, ZNF236, C18orf22, COX7A2, DDX11, TOP3A,C9orf6, UFC1, PFDN2, KLRD1, LOC643641, HSP90AB1, CLCN7, TNFAIP2, PRKCE,MRPL40, FBF1, ANKRD44, CCT5, USP40, UBXD4, LRCH1, MRPL4, SCCPDH, STX6,LOC284184, FLJ23235, GPATC3, CPSF4, CREM, HIST1H1D, HPS4, FN3KRP,ANKRD16, C8 orf16, ATF71P2, PRIC285 Prostate Serum, Prostate specificantigen (PSA) cancer saliva Urine PCA3, GOLPH2, SPINK1, TMPRSS2:ERGInfections See Table 4.2 Dental Saliva aspartate aminotransferase (AST)and alkaline caries/ phosphatase (ALP), uric acid and albumin; 12-periodontal HETE; MMP-8, TIMP-1, and ICTP disease Heavy Saliva Lead,cadmium metal poisoning Drugs/drug Saliva Marijuana, cocaine(crystalline tropane alkaloid), metabolites methamphetamine,amphetamine, heroin, methyltestosterone, mesterolone, morphine,cyclophosphamide metabolites, haloperidol, barbiturates; antipyrine,caffeine, cisplatin, cyclosporine, diazepam, digoxin, methadone,phenytoin, theophylline, tolbutamide, nicotine/cotinine, cannabisDrugs/drug Urine trichloroethanol glucuronide, anabolic steroids,metabolites androstenedione, benzodiazepines, chlordiazepoxide,lorazepam, zidovudine Allergies Saliva Allergen-specific IgAs

In other embodiments, the diagnostic marker that can be detected by thepresent method is an antibody in a sample, e.g., a diagnostic sample,that is probative for diagnosing a disease or health condition of thesubject from which the sample is derived. Table 4.4 provides a list of 5autoantibody targets, which can be used, in whole or as an epitopefragment, as a capture agent in the present method to measure the amountof the epitope-binding antibody analyte in a sample and thereby diagnosethe associated disease or health condition, e.g., an autoimmune disease.In some cases, the disease or health condition is related to an immuneresponse to an allergen. Table 4.5 provides a list of allergens, whichcan be used, in whole or as an epitope fragment, as a capture agent inthe present method to measure the amount of the epitope-binding antibodyanalyte in a sample and thereby diagnose the associated disease orhealth condition, e.g., an allergy. In certain instances, the disease orhealth condition is related to an infectious disease, where theinfectious agent may be diagnosed based on information including themeasured amount of antibodies against one or more epitopes derived fromthe infectious agent (e.g., lipopolysaccharides, toxins, proteins, etc).Table 4.6 provides a list of infectious-agent derived epitopes which canbe used, in whole or as an epitope fragment, as a capture agent in thepresent method to measure the amount of the epitope-binding antibodyanalyte in a sample and thereby diagnose the associated disease orhealth condition, e.g., an infection. Other epitopes or antigens thatmay be suitable for use in the present diagnostic method are describedin, e.g., PCT App. Pub. No. WO 2013164476, which is incorporated hereinby reference.

TABLE 4.4 Diagnostic autoantibody epitopes Disease/conditionAutoantibody Targets Cancer ACAA2; ANXA13; AQP2; ASPA; BCL2; BCL2L1;BIK; CD160; CD37; CDK4; CDK6; CHEK2; CITED2; CNN2; CTSC; CTSZ; CycE2;ELK1; FGF10; FN1; GATA3; GJA1; GNRH1; GRB2, HBB; HBE1; HIST2H2AA; HPRT1;ID2; IER2; IFI27; IFITM1; IFITM2; IL15; IL18; IL8; IL9; KRT16; LALBA;LDHA; LDHB; LECT1; MAFK; Mage3; MAGEA3; MMP2; NPPB; OAS1, p21; p53;PCNA; PENK; PEX3; PHB; PHYH; PI3; PKBα; PLN; S100A7; SCAMP1; SCGB1A1;SLC38A5; SNRP2; SNX9; SST; SSTR2; TACSTD1; TNNC2; TOB1; TSG101; VDRIP;WNT2, p62 and Koc; ZFP161, Ubiquilin-1, HOX-B6, YB-1, Osteonectin, ILF3Squamous cell protein kinase C and p53-binding protein (TP53 BP),lymphoid blast crisis lung carcinoma oncogene (LBC), Small cell lung SOXfamilies B1 and B2, MUC-1, cancer Lung cancer MUC-1, p53, surviving,LAMR1, annexin I,14-3-3-theta; AKR1B10; GOT2; HNRPR; PDIA3; NME2; RTN4;HI1FX; G3BP; HSPCA; ACTN4; PGP9.5; Colorectal cancer MUC-1, surviving,p-53; translationally controlled tumor protein; HSPC218; Ribosomalprotein S18; v-Fte-1; v-Fos transformation effector protein; MAGEA3,SSX2, NY-ESO-1, HDAC5, MBD2, TRIP4, NY-CO-45, KNSL6, HIP1R, Seb4D,KIAA1416, and LMNA; UCHL3 Hepatocellular fibrillarin and p330d/CENP-F,insulin-like growth factor II mRNA-binding carcinoma proteins (IMP) 1,IMP3 and p53, NOR-90, nucleophosmin/protein B23, cyclin B1, DNAtopoisomerase II (topo II), p62, HCC1, SG2NA, MAGE-C2, AF146731;AF219119; AF146019; Ligatin; AF220416; AF218421; AF257175; AF244135;AF243495; AF287265; AF258340; AF270491; AF286340; small nuclearRNA-associated sm-like protein; Dna J protein; CENP-F; translationallycontrolled tumor protein; LDH-A; Albumin; Hsp89αΔN; SEC63; AF100141; 14,5kDa protein; GCF2; Metallopanstimulin 1; SMP-30 D31815; Cg1 protein,;C3VS protein; F1-ATPase, β subunit; Human ribosomal protein L10;Pre-apolipoprotein CIII; Galactose-1- phosphate-uridyl-transferase(GALT); DNA polymerase Δ, small subunit; Mitochondrial DNA Renal cancerAF257175; small nuclear RNA-associated sm-like protein; Dna J protein;smooth muscle protein 22-alpha (SM22-alpha); carbonic anhydrase I (CAI)Acute leukemia Rho GDP dissociation inhibitor 2, γ-actin, F-actincapping protein (CAPZA1), heterogeneous nuclear ribonucleoprotein L(hnRNP L), tubulin-α 6, PCNA Chronic KIAA1641; PIPMT; FosB; ZNF268;SEBD4; Ikaros; p75/LDEGF; CHIP; lymphocytic PYGB; ZNF148; KIAA0336;RPL11; FMNL; HGRG8 leukemia non-Hodgkin's CENP-F, lymphoma MultipleNY-ESO-1 myeloma melanoma NY-ESO-1, MAGE-1, BAGE, GAGE, MART-1/melan A,gp100, and tyrosinase Pancreatic cancer Calreticulin, DEAD-box protein48 (DDX48) Ovarian cancer ACSBG1 , AFP, CSNK1A1 L, DHFR, MBNL1 , TP53,PRL, PSMC1 , PTGFR, PTPRA, RAB7L1 , and SCYL3, her2/neu, MUC1, c-myc,ECPKA, and NY-ESO-1, p53, UBQLN1, HOXB6, TOP2A, putative helicase-RUVBL(RUVBL), HMBA-inducible (HEXIM1), DDX5 and HDCMA Prostate cancer Bcl2,NY-ESO-1, survival protein lens epithelium-derived growth factor p75(LEDGF/p75), PRDX6/AOP2, clusterin, DJ-1, superoxide dismutase, alcoholdehydrogenase, HSP70, HSP27/HSPB1, lactoylglutathione lyase,glucose-regulated protein-78 kDa (GRP78), p62, Koc, and IMP1, α-Methylacyl-coenzyme A racemase and 5-α-reductase, AKRIA1; Brd2; C17 orf25; CAPZA1; c-MYC; Cyclin A; Cyclin B1; Cyclin D1; Drebrin; eIF4G1;HIP1; HSPA8; Lactoylglutathione lyase; MAD-CT-1; MAD-CT-2; No55; P53;P62; P90; PP4R; PIP; PSA; RPL13a; RPL22; Survivin; Syntenin 1; TDP-43;VCP; vWF; Lage-1, and Xage-1; bromo domain-containing protein 2 (BRD2),ribosomal proteins L22 and L13a, XP_373908 Breast cancer p53, c-myc,NY-ESO-1, BRCA1, BRCA2, HER2, MUC1, IGFBP-2, TOPO2α, ribosomal proteinS6, eukaryotic elongation factor 2, eukaryotic elongation factor 2kinase, and heat shock protein 90 (HSP90), Ku protein, topoisomerase I,and the 32-kDa subunit of replication protein A; CENP-F; AF146731;int-2, pentraxin I, integrin beta5, cathepsin L2 and S3 ribosomalprotein; RNA-binding protein regulatory subunit (RS), DJ-1 oncogene,glucose-6-phosphate dehydrogenase, heat shock 70-kDa protein 1 (HS71),and dihydrolipoamide dehydrogenase Nasopharyngeal MAGE, HSP70,Fibronectin, CD44, EBV antigens carcinoma Oral cancer Cyclin B1, p53Oral squamous p53 cell carcinoma Head and neck CASP-8, SART-1, TREX1, 3′repair exonuclease; BRAP (BRCA1 squamous cell associated): Nuclearlocalization protein; Trim 26 zinc finger domains; carcinoma GTF21transcription factor. Murine homolog TF11-1; NSEP1 (YB-1) transcriptionfactor; MAZ transcription factor associated with c-myc; SON (DBP-5;KIAA1019; NREBP DNA binding protein); NACA nascentpolypeptide-associated complex; NUBP2 nucleotide binding protein; EEF2Translation elongation factor 2; GU2 Putative RNA helicase; RPLI3Aribosomal protein; SFRS21P (CASP11; SIP1; SRRP1290 splicing factor);RPS12 ribosomal protein; MGC2835 RNA helicase; TMF1, TATA modulatoryfactor; PRC1 regulator of cytokinesis; KRT14 keratin 14; Viniculin;H2AFY histone family member; SLK (KIAA02304) Ste related kinase; NOL3(ARC) nuclear protein 3, apoptosis repressor; DNAJA2 member of Hsp40family; DNAJA1 member of HSP40 family; LINE-1 retrotransposon; MOG (HSPC165) Homolog of yeast protein; LIMS1 (PINCH): LIM and senescentantigen-like domain; COPB2 coatomer protein complex subunit protein;FLJ22548 hypothetical protein; C21orf97; FLJ21324; MGC15873; SSNA1Sjogrens syndrome nuclear autoantigen 1; KIAA0530, zinc finger domain;rat stannin; hypothetical protein DKFZp4340032; human FLJ23089; PC326Esophageal NY-ESO-1; SURF1, HOOK2, CENP-F, ZIC2, hCLA-iso, Ki-1/57,enigma, cancer HCA25a, SPK, LOC146223 and AGENCOURT_7565913 MetabolicGFAP syndrome/ prediabetes Diabetes Zn transporter 8, glutamic aciddecarboxylase (GAD), CD38, gad65, IA2, insulin, MRPS31, ICA1, L-typevoltage gated calcium channel; SNRPB2; DDX42; C11orf63; TCOF1; TSSK2;KDM4B; PDGFB; LTK; RPL14; VIM; GTF2I; BCL2L13; LARP6; DKFZP434K028;USP39; SERBP1; CCL19; GAD2; MCM10; ZNF688; PTEN; RP6-166C19.11; GIPC1;TIGD1; CCDC131; HTF9C; SOX5; MCF2L; TRAF3IP1; 6CKINE; ACY3; AMMECR1L;ARHGAP9; ASNS; BATF2; BMX; C9ORF25; CDC2; CHGB; CXORF38; CXORF56; DMD;ECHDC1; EIF3F; EPHA2; ERMN; FAM136A; (includes; EG: 84908); FILIP1;FLT1; GART; GIMAP6; GNG7; GTF2F1; HGS; IFI6; KDM4B; LACE1; LGALS1;LGALS7; LIMS2; LTK; LUC7L; NCAPG; (includes; EG: 64151); NME6; NUPL1;PAK4; PDE4DIP; PSIP1; RAB20; RNGTT; RPS3; SPG20; TALDO1; TBRG1; THAP1;TRAF3IP2; UBL4A; ZC3HC1; ZNF131; RAD51AP1; HADH; (HADH); C11orf16;(C11orf16); TAC3; ABR; ECE1; PPP1R2; GRINL1A; ABR; C19orf44; MUSTN1;ETHE1; BMI1; BAZ2B; ; TBC1D22A; CAMK2N2; ASS1; CCNY; MARK2; RAD51AP1;RAB38; RIOK1; HSP90AA1; C11orf74; ARID3A; LMOD1; CAPRIN1; ITGB3BP; MND1;SGK; NADK; MED9; LDHA; ARHGAP26; ANKRA2; CRY2; IL23A; DUSP14; ZBTB44;SIRT1; SLC2A3; GPR172B; CCDC89; BATF; HMOX1; ARRDC1; USF2; GBGT1; EDC3;SGIP1; GCGR; ZRANB2; NLGN4Y; GJB6; CDK10; PSG1; CCDC74A; DENND1C; MAP2K6Autoimmune cardiac troponin I (cTnI) heart disease Immunoglobulin PRKD1, MATN2, DDX17, UBE2W, CDKN1 B, SOD2, FLOT2, IQCK, A nephropathy BLZF1 ,BRD9, CDS2, EFNA3, EIF4A2, FLU, LIMCH1 , MAGEA4, MEF2D, MLLT6, MRPL28,MUTED, NKAIN4, PCTK1 , PLXNA1 , PODN, POLH, PRKD2, RNF1 1 3A, SEPT5,TNS1 , TOM1 , TRPV4, USP12, ZMYM3, CIAPIN1 , GDI2, HSPA8, SERPINA5 andTGM1 End stage renal IGLC1; IGHG1; EDC3; IGHG1; APEX2; CD3D; TRIM21;IGKV1-5; disease IGHG3; CTLA-FC; CD7; CLIP4; MAPRE1; SNRPB2; IGHG1;ZBTB44; CD3D; IGHG1; TRAM1; ERR beta-; LBD; CNBP; OLFM1; IGHM; SIRT5;CEP290; PHLDA1 Glomerular nephritis Addison's disease 21-hydroxylase,P450-17α-hydroxylase (17OH) and P450-side chain cleavage (SCC) Primaryovarian Jo-1, proteinase 3 (PR3) insufficiency Sjögren's IgA, IgG, IgMautoantibodies; IgA, lactoferrin and beta2-microglobulin; syndromelysozyme C, and cystatin C, amylase and carbonic anhydrase SSA/Ro;LA/SS-B Systemic lupus CDC25B, APOBEC3G, ARAF, BCL2A1, CLK1, CREB1,CSNK1G1, erythematosus CSNK2A1, CWC27, DLX4, DPPA2, EFHD2, EGR2, ERCC2,EWSR1, (SLE) EZH2, FES, FOS, FTHL17, GEM, GNA15, GNG4, HMGB2, HNRNPUL1,HOXB6, ID2, IFI35, IGF2BP3, IGHG1, JUNB, KLF6, LGALS7, LIN28A, MLLT3,NFIL3, NRBF2, PABPC1, PATZ1, PCGF2, PPP2CB, PPP3CC, PRM1, PTK2, PTPN4,PYGB, RET, RPL18A, RPS7, RRAS, SCEL, SH2B1, SMAD2, STAM, TAF9, TIE1,UBA3, VAV1, WT1, ZAP70, or ZNRD1 KIT, C6orf93, RPL34, DOM3Z, COPG2,DNCL12, RRP41; FBXO9; RALBP1, PIAS2; EEF1D; CONI; KATNB1; POLR2E; CCT3;KIAA0643; RPL37A, GTF2H2; MAP2K5; CDK3; RPS6KA1; MARK4, MTO1; MGC42105;NFE2L2; WDR45L, STK4, PFKFB3; NTRK3; MLF1; TRIM37, ACTL7B, RPL18A,CKS1B; TUBA1, NME6, SUCLA2, IGHG1, PRKCBP1; BAG3; TCEB3; RPL15, SSX4;MAP2K7; EEF1G; RNF38, PHLDA2, KCMF1; NUBP2, VPS45A SSA/Ro; dsDNA; Smith;histones; thrombin; v-Fos transformation effector protein, tryptase, Smantigen, beta 2; cardiolipin; glycoprotein I β2; EndothelialPC/activated PC receptor; human gamma enolase CREST syndrome centromereSystemic Type I topoisomerase sclerosis Primary biliary nucleoporin 62,Sp100 nuclear antigen, nucleoporin 210kDa, mitochondria, cirrhosismitochondrial pyruvate dehydrogenase (PDH) or E3 binding proteinDermatitis eTG herpetiformis Miller-Fisher ganglioside GQ1B SyndromeWegener's c-ANCA granulomatosis Neuropathies ganglioside GD3,ganglioside GM1, GA1, GM2, MAG microscopic p-ANCA poly angiitisPolymyositis Signal recognition particles scleromyositis exosome complexSignal recognition particles myasthenia gravis nicotinic acetylcholinereceptor Signal recognition particles, muscle-specific kinase (MUSK)Signal recognition particles Lambert-Eaton voltage-gated calcium channel(P/Q-type) myasthenic syndrome Hashimoto's thyroid peroxidasethyroiditis Graves' disease TSH receptor paraneoplastic Hu, Yo(cerebellar Purkinje Cells), amphiphysin cerebellar syndromeencephalitis voltage-gated potassium channel (VGKC),N-methyl-D-aspartate receptor (NMDA) Sydenham's basal ganglia neuronschorea antiphospholipid glycoprotein 1 ( 2GPl), Endothelial PC/activatedPC receptor syndrome Systemic proteinase 3 (PR3) and myeloperoxidase(MPO) vasculitis Neuromyelitis aquaporin-4 Allergies Allergen-specificIgAs Rheumatoid Rheumatoid factor, cyclic citrullinated protein; humancartilage gp39 arthritis peptides and type II collagen; citrullinatedfibrinogen, citrullinated vimentin, citrulline-substituted filaggrinpeptides, hnRNP-A2/B1, BiP, tryptase Asthma tryptase Multiple sclerosismyelin basic protein, spectrin, fodrin, myelin oligodentrocyteglycoprotein, proteolipid protein (PLP), 2′, 3′-cyclicnucleotide-phosphodiesterase (CNP), Glc(α1, 4)Glc(α) (GAGA4), Glc(α1,6)Glc(α) (GAGA6) amyotrophic HMGB1 lateral sclerosis (ALS) Idiopathicplatelet glycoprotein (GP) IIb/IIIa, GPIb/IX, GPIa/IIa thrombocytopenicpurpura Thrombosis thrombomodulin Cardiovascular EndothelialPC/activated PC receptor; IL-1alpha, alpha-actinin-2 (aActn2); diseasealpha-Myosin Heavy Chain (alpha-MHC-S 1); SI fragment of alpha-MyosinHeavy Chain 6 (alpha-MHC6-Sl); alpha-Myosin Heavy Chain 7 (MyHC7)post-streptococcal ELAVL2, ELAVL3, ELAVL4, Nova-1, Nova-2, Cdr1, Cdr2;and Cdr3 disease such as PANDAS, post- GABHS glomerulonephritis,rheumatic fever, autism and Syndenham's chorea Parkinson'salpha-synuclein; myelin basic protein (MBP), proteolipid protein (PLP),Disease myelin oligodendrocyte glycoprotein (MOG), myelin associatedglycoprotein (MAG), oligodendrocytes specific protein (OSP) perniciousanemia Vitamin B₁₂

TABLE 4.5 Allergen epitopes Source Allergen mites Acas13, Blot1, Blot3,Blot4, Blot5, Blot6, Blot10, Blot11, Blot12, Blot13, Blot19; Americanhouse dust mite (Derf1, Derf2, Derf3, Derf7, Derf10, Derf11, Derf14,Derf15, Derf16, Derf17, Derf18w); house dust mite (Derm1); Europeanhouse dust mite (Derp1, Derp2, Derp3, Derp4, Derp5, Derp6, Derp7, Derp8,Derp9, Derp10, Derp11, Derp14, Derp20, Derp21); mite (Eurm2; Eurm14);storage mite (Glyd2, Lepd2, Lepd5, Lepd7, Lepd10, Lepd13, Tyrp2,Tyrp13); Dermatophagoides farinae (Derf1.0101, Derf1.0102, Derf1.0103,Derf1.0104, Derf1.0105, Derf2.0101, Derf2.0102, Derf2.0103, Derf2.0104,Derf2.0105, Derf2.0106, Derf2.0107, Derf2.0108, Derf2.0109, Derf2.0110,Derf2.0111, Derf2.0112, Derf2.0113, Derf2.0114, Derf2.0115, Derf2.0116,Derf2.0117); Dermatophagoides pteronyssinus (Derp1.0101, Derp1.0102,Derp1.0103, Derp1.0104, Derp1.0105, Derp1.0106, Derp1.0107, Derp1.0108,Derp1.0109, Derp1.0110, Derp1.0111, Derp1.0112, Derp1.0113, Derp1.0114,Derp1.0115, Derp1.0116, Derp1.0117, Derp1.0118, Derp1.0119, Derp1.0120,Derp1.0121, Derp1.0122, Derp1.0123, Derp2.0101, Derp2.0102, Derp2.0103,Derp2.0104, Derp2.0105, Derp2.0106, Derp2.0107, Derp2.0108, Derp2.0109,Derp2.0110, Derp2.0111, Derp2.0112, Derp2.0113); Euroglyphus maynei(Eurm2.0101, Eurm2.0102); Glycyphagus domesticus (Glyd2.0101,Glyd2.0201); and Lepidoglyphus destructor (Lepd2.0101, Lepd2.0101,Lepd2.0101, Lepd2.0102, Lepd2.0201, Lepd2.0202) Pollen Short Ragweed(Ambrosia artemisiifolia) allergen, Amb a 1, Amba2, Amba3, Amba5, Amba6,Amba7, Amba8, Amba9, Amba10; Betula verrucosa allergen, Bet v 1, Phleumpratense allergen, Phl p 5), giant ragweed (Ambt5); mugwort (Artv1,Artv2, Artv3, Artv4, Artv5, Artv6); sunflower (Hela1, Hela2, Hela3);Mercurialis annua (Mera1); lamb’s-quarters, pigweed (Chea1); whitegoosefoot (Chea2, Chea3); Russian- thistle (Saik1); Rosy periwinkle(Catr1); English plantain (Plal1); Japanese hop (Humj1); Parietariajudaica (Parj1, Parj2, Parj3); Parietaria officinalis (Paro1); Ambrosiaartemisiifolia (Amba8.0101, Amba8.0102, Amba9.0101, Amba9.0102);Plantago lanceolata (Plal1.0101, Plal1.0102, Plal1.0103); and Parietariajudaica (Parj1.0101, Parj1.0102, Parj1.0201, Par2.0101, Parj2.0102,Parj3.0101, Parj3.0102), Bermuda grass (Cynd1, Cynd7, Cynd12, Cynd15,Cynd22w, Cynd23, Cynd24); orchard grass (Dacg1, Dacg2, Dacg3, Dacg5);meadow fescue (Fesp4w); velvet grass (Holl1); rye grass (Lolp1, Lolp2,Lolp3, Lolp5, Lolp11); canary grass (Phaa1); Timothy (Phlp1, Phlp2,Phlp4, Phlp5, Phlp6, Phlp11, Phlp12, Phlp13); Kentucky blue grass(Poap1, Poap5); Johnson grass (Sorh1); Cynodon dactylon (Cynd1.0101,Cynd1.0102, Cynd1.0103, Cynd1.0104, Cynd1.0105, Cynd1.0106, Cynd1.0107,Cynd1.0201, Cynd1.0202, Cynd1.0203, Cynd1.0204); Holcus lanatus (Holl1.0101, Holl1.0102); Lolium perenne (Lolp1.0101, Lolp1.0102, Lolp1.0103,Lolp5.0101, Lolp5.0102); Phleum pretense (Phlp1.0101, Phlp1.0102,Phlp4.0101, Phlp4.0201, Phlp5.0101, Phlp5.0102, Phlp5.0103, Phlp5.0104,Phlp5.0105, Phlp5.0106, Phlp5.0107, Phlp5.0108, Phlp5.0201, Phlp5.0202);and Secale cereale (Secc20.0101, Secc20.0201), Alder (Alng1); Birch(Betv1, Betv2, Betv3, Betv4, Betv6, Betv7); hornbeam (Carb1); chestnut(Cass1, Cass5, Cass8); hazel (Cora1, Cora2, Cora8, Cora9, Cora10,Cora11); White oak (Quea1); Ash (Frae1); privet (Ligv1); olive (Olee1,Olee2, Olee3, Olee4, Olee5, Olee6, Olee7, Olee8, Olee9, Olee10); Lilac(Syrv1); Sugi (Cryj1, Cryj2); cypress (Cupa1); common cypress (Cups1,Cups3w); mountain cedar (Juna1, Juna2, Juna3); prickly juniper (Juno4);mountain cedar (Juns1); eastern red cedar (Junv1); London plane tree(Plaa1, Plaa2, Plaa3); date palm (Phod2); Betula verrucosa (Betv1.0101,Betv1.0102, Betv1.0103, Betv1.0201, Betv1.0301, Betv1.0401, Betv1.0402,Betv1.0501, Betv1.0601, Betv1.0602, Betv1.0701, Betv1.0801, Betv1.0901,Betv1. 1001, Betv1.1101, Betv1.1201, Betv1.1301, Betv1.1401, Betv1.1402,Betv1.1501, Betv1.1502, Betv1.1601, Betv1.1701, Betv1.1801, Betv1.1901,Betv1.2001, Betv1.2101, Betv1.2201, Betv1.2301, Betv1.2401, Betv1.2501,Betv1.2601, Betv1.2701, Betv1.2801, Betv1.2901, Betv1.3001, Betv1.3101,Betv6.0101, Betv6.0102); Carpinus betulus (Carb1.0101, Carb1.0102,Carb1.0103, Carb1.0104, Carb1.0105, Carb1.0106, Carb1.0106, Carb1.0106,Carb1.0106, Carb1.0107, Carb1.0107, Carb1.0108, Carb1.0201, Carb1.0301,Carb1.0302); Corylus avellana (Cora1.0101, Cora1.0102, Cora1.0103,Cora1.0104, Cora1.0201, Cora1.0301, Cora1.0401, Cora1.0402, Cora1.0403,Cora1.0404); Ligustrum vulgare (Ligv1.0101, Ligv1.01.02); Olea europea(Olee1.0101, Olee1.0102, Olee1.0103, Olee1.0104, Olee1.0105, Olee1.0106,Olee1.0107); Syringa vulgaris (Syrv1.0101, Syrv1.0102, Syrv1.0103);Cryptomeria japonica (Cryj2.0101, Cryj2.0102); and Cupressussempervirens (Cups1.0101, Cups1.0102, Cups1.0103, Cups1.0104,Cups1.0105) mold Alternaria alternata allergen, Alt a 1, Alta3, Alta4,Alta5, Alta6, Alta7, Alta8, Alta10, Alta12, Alta13, Aspergillusfumigatusallergen, Asp f 1, Aspf2, Aspf3, Aspf4, Aspf5, Aspf6, Aspf7, Aspf8,Aspf9, Aspf10, Aspf11, Aspf12, Aspf13, Aspf15, Aspf16, Aspf17, Aspf18,Aspf22w, Aspf23, Aspf27, Aspf28, Aspf29); Aspergillus niger (Aspn14,Aspn18, Aspn25); Aspergillus oryzae (Aspo13, Aspo21); Penicilliumbrevicompactum (Penbl3, Penb26); Penicillium chrysogenum (Pench13,Pench18, Pench20); Penicillium citrinum (Penc3, Penc13, Penc19, Penc22w,Penc24); Penicillium oxalicum (Penol8); Fusarium culmorum (Fuse1,Fusc2); Trichophyton rubrum (Trir2, Trir4); Trichophyton tonsurans(Trit1, Trit4); Candida albicans (Canda1, Canda3); Candida boidinii(Candb2); Psilocybe cubensis (Psic1, Psic2); shaggy cap (Cope1, Copc2,Copc3, Copc5, Copc7); Rhodotorula mucilaginosa (Rhom1, Rhom2);Malassezia furfur (Malaf2, Malaf3, Malaf4); Malassezia sympodialis(Malas1, Malas5, Malas6, Malas7, Malas8, Malas9, Malas10, Malas11,Malas12, Malas13); Epicoccum purpurascens (Epip1); and Alternariaalternate (Alta1.0101, Alta1.0102), Aspergillus versicolor antigen, S.chartarum antigen), Cladosporium herbarum (Clah2, Clah5, Clah6, Clah7,Clah8, Clah9, Clah10, Clah12); Aspergillus flavus (Aspf113); mammals Bosdomesticus dander allergen, Bos d 2, Bosd3, Bosd4, Bosd5, Bosd6, Bosd7,Bosd8, Bosd2.0101, Bosd2.0102, Bosd2.0103, Canis familiaris allergen,Can f 1,Canf2, Canf3, Canf4, Equus caballus allergen, Equc1, Equc2,Equc3, Equc4, Equc5, Felis domesticus allergen, Fel d 1, Feld2, Feld3,Feld4, Feld5w, Feld6w, Feld7w, guinea pig (Cavp1, Cavp2); Mouse UrinaryProtein (MUP, Musm1) allergen, Mus m 1, Rat Urinary Protein (RUP, Ratn1)allergen, Rat n 1., Equus caballus (Equc2.0101, Equc2.0102)) InsectsMosquito (Aeda1, Aeda2); honey bee (Apim1, Apim2, Apim4, Apim6, Apim7);bumble bee (Bomp1, Bomp4); German cockroach (Blag1, Blag2, Blag4, Blag5,Blag6, Blag7, Blag8); American cockroach (Pera1, Pera3, Pera6, Pera7);midge (Chit1-9, Chit1.01, Chit1.02, Chit2.0101, Chit2.0102, Chit3,Chit4, Chit5, Chit6.01, Chit6.02, Chit7, Chit8, Chit9); cat flea (Ctef1,Ctef2, Ctef3); pine processionary moth (Thap1); silverfish (Leps1);white face hornet (Dolm1, Dolm2, Dolm5); yellow hornet (Dola5); wasp(Pola1, Pola2, Pola5, Pole1, Pole5, Polf5, Polg5, Polm5, Vesvi5);Mediterranean paper wasp (Pold1, Pold4, Pold5); European hornet (Vespc1,Vespc5); giant asian hornet (Vespm1, Vespm5); yellowjacket (Vesf5,Vesg5, Vesm1, Vesm2, Vesm5, Vesp5, Vess5, Vesv1, Vesv2, Vesv5);Australian jumper ant (Myrp1, Myrp2); tropical fire ant (Solg2, Solg4);fire ant (Soli2, Soli3, Soli4); Brazilian fire ant (Sols2); Californiakissing bug (Triap1); Blattella germanica (Blag1.0101, Blag1.0102,Blag1.0103, Blag1.02, Blag6.0101, Blag6.0201, Blag6.0301); PeriplanetaAmericana (Pera1.0101, Pera1.0102, Pera1.0103, Pera1.0104, Pera1.02,Pera3.01, Pera3.0201, Pera3.0202, Pera3.0203, Pera7.0101, Pera7.0102);Vespa crabo (Vespc5.0101, Vespc5.0101); and Vespa mandarina (Vesp m1.01, Vesp m 1.02) Rubber rubber (latex)(Hevb1, Hevb2, Hevb3, Hevb4,Hevb5, Hevb6.01, Hevb6.02, Hevb6.03, Hevb7.01, Hevb7.02, Hevb8, Hevb9,Hevb10, Hevb11, Hevb12, Hevb13); Hevea brasiliensis (Hevb6.01,Hevb6.0201, Hevb6.0202, Hevb6.03, Hevb8.0101, Hevb8.0102, Hevb8.0201,Hevb8.0202, Hevb8.0203, Hevb8.0204, Hevb10.0101, Hevb10.0102,Hevb10.0103, Hevb11.0101, Hevb11.0102) Others Nematode (Anis1, Anis2,Anis3, Anis4); pigeon tick (Argr1); worm (Ascs1); papaya (Carp1); softcoral (Denn1); human autoallergens (Homs1, Homs2, Homs3, Homs4, Homs5);obeche (Trips1)

TABLE 4.6 Infectious agent-derived epitopes Infectious Agent EpitopeMycobacterium tuberculosis isocitrate dehydrogenase (ICDs) Influenzavirus Hemagglutinin (H1), neuraminidase (N1) Dengue virus envelope (E)Toxoplasma gondii microneme proteins, SAG1, SAG2, GRA1, GRA2, GRA4,GRA6, GRA7, GRA3, ROP1, ROP2, p30, MIC3, MIC2, M2AP, p29, p35, p66Entamoeba histolytica M17, neutral thiol proteinase Streptococcuspneumonia Pneumolysin, pneumococcal histidine triad D (PhtD),pneumococcal choline-binding protein A (PepA), pneumococcal histidinetriad E (PhtE), LytB Mycoplasma pneumonia exotoxin Epstein-Barr virusVCA Helicobacter pylori CagA, Vacuolating protein, ureB, hsp60, ureH,urea, ferritin like protein Campylobacter jejuni PEB1, PEB3 Bacillusanthracis SAP SARS virus RNA-dependent replicases Ia and Ib, spike (S)protein, small envelope (E) protein, membrane (M) protein, andnucleocapsid (N) protein Ebola virus Nucleoprotein N Schmallenberg virusN nucleoprotein enterovirus 71 VPl protein Japanese Encephalitis virussoluble E protein, envelope E protein Ross River virus soluble E2protein Mayaro virus soluble E2 protein Equine Encephalitis virusessoluble E2 protein Akabane virus N nucleoprotein Infectious AgentEpitope human betacoronavirus Nucleoprotein N, protein S Hepatitis Cvirus protein C, core antigen Hepatitis E virus protein C Plasmodiumfalciparum MSP-1 + AMA-1 protein Leptospira interrogans HbpA, LruA,LruB, or LipL32

In some embodiments, the devices, systems and methods of the inventioncan be used to detect a diagnostic marker that is a microRNA (miRNA)biomarker associated with a disease or a health condition. Table 4.7provides an exemplary list of miRNA biomarkers that can be used andtheir associated diseases/health conditions.

TABLE 4.7 Diagnostic miRNA markers Disease/Condition Marker* Breastcancer miR-10b, miR-21, miR-125b, miR-145, miR-155, miR-191, miR-382,MiR-1, miR-133a, miR-133b, miR-202, miR-1255a, miR-671-3p, miR- 1827,miR-222, miR-744, miR-4306, miR-151-3p, miR-130, miR-149, miR-652,miR-320d, miR-18a, miR-181a, miR-3136, miR-629, miR- 195, miR-122,miR-375, miR-184, miR-1299, miR381, miR-1246, miR- 410, miR-196a,miR-429, miR-141, miR-376a, miR-370, miR-200b, miR-125a-5p, miR-205,miR-200a, miR-224, miR-494, miR-216a, miR- 654-5p, miR-217, miR-99b,miR-885-3p, miR-1228, miR-483-5p, miR- 200c, miR-3065-5p, miR-203,miR-1308, let-7a, miR-17-92, miR-34a, miR-223, miR-150, miR-15b,miR-199a-5p, miR-33a, miR-423-5p, miR- 424, let-7d, miR-103, miR-23b,miR-30d, miR-425, miR-23a, miR-26a, miR-339-3p, miR-127-3p, miR-148b,miR-376a, miR-376c, miR-409-3p, miR-652, miR- 801 (miR-92a, miR-548d-5p,miR-760, miR-1234, miR-18b, miR-605, miR- 193b, miR-29) Leukemia miR-98,miR-155, miR-21, let-7, miR-126, miR-196b, miR-128, miR- 195, miR-29a,miR-222, miR-20a, miR-150, miR-451, miR-135a, miR- 486-5p, miR-92,miR-148a, miR-181a, miR-20a, miR-221, miR-625, miR-99b (miR-92a, miR-15,miR-16, miR-15a, miR-16-1, miR-29) Multiple myeloma miR-15a, miR-16,miR-193b-365, miR-720, miR-1308, miR-1246, miR- 1, miR-133a, miR-221,miR-99b, Let-7e, miR-125a-5p, miR-21, miR- 181a/b, miR-106b-25, miR-32,miR-19a/b, miR-17-92, miR-17, miR-20, miR-92, miR-20a, miR-148a,miR-153, miR-490, miR-455, miR-642, miR-500, miR-296, miR-548d, miR-373,miR-554, miR-888, miR-203, miR-342, miR-631, miR-200a, miR-34c, miR-361,miR-9*, miR-200b, miR-9, miR-151, miR-218, miR-28-3p, miR-200c, miR-378,miR-548d- 5p, miR-621, miR-140-5p, miR-634, miR-616, miR-130a, miR-593,miR-708, miR-200a*, miR-340, miR-760, miR-188-5p, miR-760, miR- 885-3p,miR-590-3p, miR-885-5p, miR-7, miR-338, miR-222, miR-99a, miR-891a,miR-452, miR-98, miR-629, miR-515-3p, miR-192, miR-454, miR-151-3p,miR-141, miR-128b, miR-1227, miR-128a, miR-205, miR- 27b, miR-608,miR-432, miR-220, miR-135a, miR-34a, miR-28, miR- 412, miR-877,miR-628-5p, miR-532-3p, miR-625, miR-34b, miR-31, miR-106b, miR-146a,miR-210, miR-499-5p, miR-140, miR-188, miR- 610, miR-27a, miR-142-5p,miR-603, miR-660, miR-649, miR-140-3p, miR-300, miR-335, miR-206,miR-20b, miR-130b, miR-183, miR-652, miR-133b, miR-191, miR-212,miR-194, miR-10Om miR-1234m miR- 182m miR-888, miR-30e-5p, miR-574,miR-135b, miR-125b, miR-502m miR-320, miR548-421, miR-129-3p, miR-190b,miR-18a, miR-549, 338- 5p, miR-756-3p, miR-133a, miR-521, miR-486-3p,miR-553, miR-452*, miR-628-3p, miR-620, miR-566, miR-892a, miR-miR-339-5p, miR- 628, miR-520d-5p, miR-297, miR-213, miR-519e*,miR-422a, miR-198, miR-122a, miR-1236, miR-548c-5p, miR-191*, miR-583,miR-376c, miR-34c-3p, miR-453, miR-509, miR-124a, miR-505, miR-208, miR-659, miR-146b, miR-518c, miR-665, miR-324-5p, miR-152, miR-548d,miR-455-3p (miR-15a, miR-373*, miR-378*, miR-143, miR-337, miR-223, miR-369-3p, miR-520g, miR-485-5p, miR-524, miR-520h, miR-516-3p, miR- 519d,miR-371-3p, miR-455, miR-520b, miR-518d, miR-624, miR-296, miR-16)monoclonal miR-21, miR-210, miR-9*, miR-200b, miR-222, miR-376gammopathy of (miR-339, miR-328) undetermined significanceMyelodisplastic (Let-7a, miR-16) syndrome Lymphoma miR-155, miR-210,miR-21, miR-17-92, miR-18a, miR-181a, miR-222, miR-20a/b, miR-194,miR-29, miR-150, miR-155, miR-223, miR-221, let-7f, miR-146a, miR-15,miR-16-1, miR-34b/c, miR-17-5p (miR-20b, miR-184, miR-200a/b/c, miR-205,miR-34a, miR-29a, miR- 29b-1, miR-139, miR-345, miR-125a, miR-126,miR-26a/b, miR-92a, miR-20a, miR-16, miR-101, miR-29c miR-138, miR-181b)Lung cancer let-7c, miR-100, miR-10a, miR-10b, miR-122a, miR-125b,miR-129, miR-148a, miR-150, miR-17-5p, miR-183, miR-18a*, miR-18b, miR-190, miR-192, miR-193 a, miR-196b, miR-197, miR-19a, miR-19b, miR- 200c,miR-203, miR-206, miR-20b, miR-210, miR-214, miR-218, miR- 296,miR-30a-3p, miR-31, miR-346, miR-34c, miR-375, miR-383, miR- 422a,miR-429, miR-448, miR-449, miR-452, miR-483, miR-486, miR- 489, miR-497,miR-500, miR-501, miR-507, miR-511, miR-514, miR- 516-3p, miR-520d,miR-527, miR-7, miR-92, miR-93, miR-99a, miR-25, miR-223, miR-21,miR-155, miR-556, miR-550, miR-939, miR-616*, miR-146b-3p andmiR-30c-l*, miR-142-5p, miR-328, miR-127, miR- 151, miR-451, miR-126,miR-425-5p, miR-222, miR-769-5p, miR-642, miR-202, miR-34a (let-7a,let-7d, let-7e, let-7g, let-7i, miR-1, miR-103, miR-106a, miR- 125a,miR-130a, miR-130b, miR-133a, miR-145, miR-148b, miR-15a, miR-15b,miR-17-3p, miR-181d, miR-18a, miR-196a, miR-198, miR- 199a, miR-199a*,miR-212, miR-22, miR-221, miR-23a, miR-23b, miR- 26a, miR-27a, miR-27b,miR-29b, miR-30b, miR-30d, miR-30e-3p, miR-320, miR-323, miR-326,miR-331, miR-335, miR-339, miR-374, miR-377, miR-379, miR-410, miR-423,miR-433, miR-485-3p, miR- 485-5p, miR-487b, miR-490, miR-491, miR-493,miR-493-3p, miR-494, miR-496, miR-502, miR-505, miR-519d, miR-539,miR-542-3p, miR- 98) Colorectal cancer miR-29a, miR-17-3p, miR-92,miR-21, miR-31, miR-155, miR-92a, miR-141, mir-202, mir-497, mir-3065,mir-450a-2, mir-3154, mir-585, mir-3175, mir-1224, mir-3117, mir-1286(miR-34) Prostate cancer miR-141, miR-375, miR-16, miR-92a, miR-103,miR-107, miR-197, miR-485-3p, miR-486-5p, miR-26a, miR-92b, miR-574-3p,miR-636, miR-640, miR-766, miR-885-5p, miR-141, miR-195, miR-375,miR-298, miR-346, miR-1-1, miR-1181, miR-1291, miR-133a-1, miR-133b,miR- 1469, miR-148*, miR-153, miR-182, miR-182*, miR-183, miR- 183*,miR-185, miR-191, miR-192, miR-1973, miR-200b, miR-205, miR-210,miR-33b*, miR-3607-5p, miR-3621, miR-378a, miR-429, miR-494, miR-582,miR-602, miR-665, miR-96, miR-99b*, miR-100, miR-125b, miR-143,miR-200a, miR-200c, miR-222, miR-296, and miR-425-5p Ovarian cancermiR-21, miR-92, miR-93, miR-126, miR-29a, miR-141, miR-200a/b/c,miR-203, miR-205, miR-214, miR-221, miR-222, miR- 146a, miR-150, miR-193a-5p, miR-31, miR-370, let-7d, miR-508-5p, miR-152, miR- 509-3-5p,miR-508-3p, miR-708, miR-431, miR-185, miR-124, miR- 886-3p,hsa-miR-449, hsa-miR-135a, hsa-miR-429, miR-205, miR-20b,hsa-miR-142-5p, miR-29c, miR-182 (miR-155, miR-127, miR-99b) Cervicalcancer miR-21, miR-9, miR-200a, miR-497 (miR-143, miR-203, miR-218)Esophageal miR-21, hsa-miR-200a, hsa-miR-345, hsa-miR-373*, hsa-miR-630,hsa- carcinoma miR-663, hsa-miR-765, hsa-miR-625, hsa-miR-93,hsa-miR-106b, hsa- miR-155, hsa-miR-130b, hsa-miR-30a, hsa-miR-301a,hsa-miR-15b (miR-375) Gastric cancer miR-17-5p, miR-21, miR-106a,miR-106b, miR-187, miR-371-5p, miR- 378 (let-7a, miR-31, miR-192,miR-215, miR-200/141) Pancreatic cancer, miR-210, miR-21, miR-155,miR-196a, miR-1290, miR-20a, miR-24, ductal miR-25, miR-99a, miR-185,miR-191, miR-18a, miR-642b-3p, miR-885- adenocarcinoma 5p, miR-22-3p,miR-675, miR-212, miR-148a*, miR-148, miR-187, let- 7g*, miR-205,miR-944, miR-431, miR-194*, miR-769-5p, miR-450b- 5p, miR-222, miR-222*,miR-146, miR-23a*, miR-143*, miR-216a, miR-891a, miR-409-5p, miR-449b,miR-330-5p, miR-29a*, miR-625 Hepatocellular miR-500, miR-15b, miR-21,miR-130b, miR-183, miR-122, miR-34a, carcinoma miR-16, miR-221, miR-222Melanoma miR-150, miR-15b, miR-199a-5p, miR-33a, miR-423-5p, miR-424,miR- let-7d, miR-103, miR-23b, miR-30d, miR-425, miR-222, miR-23a, miR-26a, miR-339-3p Squamous cell miR-184a carcinoma Bladder cancer miR-126,miR-182 (urine), miR-16, miR-320 (miR-143, miR-145, miR-200/141) Renalcancer miR-1233, miR-199b-5p, miR-130b (miR-10b, miR-139-5p) Oral cancermiR-31, miR-24, miR-184; miR-34c; miR-137; miR-372; miR-124a; miR-21;miR-124b; miR-31; miR-128a; miR-34b; miR-154; miR-197; miR-132; miR-147;miR-325; miR-181c; miR-198; miR-155; miR-30a- 3p; miR-338; miR-17-5p;miR-104; miR-134; miR-213 (miR-200a, miR-125a, miR-133a; miR-99a;miR-194; miR-133; miR- 219; miR-100; miR-125; miR-26b; miR-138; miR-149;miR-195; miR- 107; and miR-139 (saliva)) Head and neck miR-455-3p,miR-455-5p, miR-130b, miR-130b*, miR-801, miR-196a, cancer miR-21,miR-31 Endometrial cancer miR-503, miR-424, miR-29b, miR-146a, miR-31Testicular cancer miR-372, miR-373 Glioblastoma miR-21, miR-221, miR-222Thyroid cancer miR-187, miR-221, miR-222, miR-146b, miR-155, miR-224,miR-197, miR-192, miR-328, miR-346, miR-512-3p, miR-886-5p, miR-450a,miR-301 b, miR-429, miR-542-3p, miR-130a, miR-146b-5p, miR-199a- 5p,miR-193a-3p, miR-152, miR-199a-3p/miR-199b-3p, miR-424, miR- 22,miR-146a, miR-339-3p, miR-365, let-7i*, miR-363*, miR-148a, miR-299-3p,let-7a*, miR-200b, miR-200c, miR-375, miR-451, miR- 144, let-7i,miR-1826, miR-1201, miR-140-5p, miR-126, miR-126*, let- 7f-2*, miR-148b,miR-21 *, miR-342- 3p, miR-27a, miR-145*, miR- 513b, miR-101, miR-26a,miR-24, miR-30a*, miR-377, miR-518e7, miR-519a7, miR-519b-5p,miR-519c-5p, miR-5227, miR-523*, miR- 222*, miR-452, miR-665, miR-584,miR-492, miR-744, miR-662, miR- 219-2-3p, miR-631 and miR-637,miRPIus-E1078, miR-19a, miR-501- 3p, miR-17, miR-335, miR-106b, miR-15a,miR-16, miR-374a, miR- 542-5p, miR-503, miR-320a, miR-326, miR-330-3p,miR-1, miR-7b, miR-26b, miR-106a, miR-13 9, miR-141, miR-143, miR- 149,miR-182, miR-190b, miR-193a, miR-193b, miR-211, miR-214, miR-218,miR-302c*, miR-320, miR-324, miR-338, miR-342, miR-367, miR-378,miR-409, miR-432, miR-483, miR-486, miR-497, miR-518f, miR-574, miR-616,miR-628, miR-663b, miR-888, miR-1247, miR- 1248, miR-1262, and miR-1305miR-21, miR-25, miR-32, miR-99b*, miR-125a, miR-125b, miR-138, miR-140,miR-181a, miR-213, miR-221, miR-222, and miR-345 Ischemic heart miR-1,miR-30c, miR-133, miR-145, miR-208a/b, miR-499, miR-663b,disease/Myocardial miR-1291 infarction (miR-126, miR-197, miR-223) Heartfailure miR-29b, miR-122, miR-142-3p, miR-423-5p, miR-152, miR-155, miR-497 (miR-107, miR-125b, miR-126, miR-139, miR-142-5p, miR-497) StrokemiR-124, miR-145 (miR-210) Coronary artery miR-21, miR-27b, miR-130a,miR-134, miR-135a, miR-198, miR-210, disease miR-370 (miR-17, miR-92a,miR-126, miR-145m miR-155m miR-181a, miR-221, miR-222) Diabetes miR-9,miR-28-3p, miR-29a, miR-30d, miR-34a, miR-124a, miR-146a, miR-375,miR-503, 144 (miR-15a, miR-20b, miR-21, miR-24, miR-126, miR-191,miR-197, 223, miR-320, miR-486) Hypertension Hcmv-miR-UL112, Let-7e(miR-296-5p) Chronic HCV miR-155, miR-122, miR-125b, miR-146a, miR-21infection Liver injury miR-122, miR-192 Sepsis miR-146a, miR223Arthritis miR-125a-5p, miR-24, miR-26a, miR-9, miR-25, miR-98, miR-146a,miR-124a, miR-346, miR-223, miR-155 (miR-132, miR-146) Systemic lupus(miR-200a/b/c, miR-205, miR-429, miR-192, miR-141, miR-429, miR-erythematosus 192 (urine or serum)) Chron disease miR-199a-5p,miR-362-3p, miR-532-3p, miR-plus-E1271, miR-340* (miR-149*,miR-plus-F1065) Ulcerative colitis miR-28-5p, miR-151-5p, miR-199-5p,miR-340*, miR-plus-E1271, miR- 103-2*, miR-362-3p, miR-532-3p (miR-505)Asthma miR-705, miR-575, let-7d, miR-173p, miR-423-5p, miR-611, miR-674,let-7f-1, miR-23b, miR-223, miR-142-3p, let-7c, miR-25, miR-15b, let-7g, and miR-542-5p, miR-370 (miR-325, miR-134, miR-198, miR-721,miR-515-3p, miR-680, miR- 601, miR-206, miR-202, miR-671, miR-381,miR-630, miR-759, miR- 564, miR-709, miR-513, miR-298) Chronic pulmonarymiR-148a, miR-148b, miR-152 disease Idiopathic miR-199a-5p pulmonaryfibrosis Alzheimer’s disease (miR-137, miR-181c, miR-9, miR-29a/b)Duchenne muscular miR-1, miR-133a, miR-206 dystrophy Multiple sclerosismiR-633, miR-181c-5p (CSF), miR-17-5p, miR-193a, miR-326, miR- 650,miR-155, miR-142-3p, miR-146a, miR-146b, miR-34a, miR-21, miR-23a,miR-199a, miR-27a, miR-142-5p, miR-193a, miR-15a, miR- 200c, miR-130a,miR-223, miR-22, miR-320, miR-214, miR-629, miR- 148a, miR-28, miR-195,miR-135a, miR-204, miR-660, miR-152, miR- 30a-5p, miR-30a-3p, miR-365,miR-532, let-7c, miR-20b, miR-30d, miR-9, hsa-mir-18b, hsa-mir-493,hsa-mir-599, hsa-mir-96, hsa-mir-193, hsa-mir-328, hsa-mir-409-5p,hsa-mir-449b, hsa-mir-485-3p, hsa-mir- 554 (miR-922 (CSF), miR-497,miR-1 and miR-126, miR-656, miR-184, miR-139, miR-23b, miR-487b,miR-181c, miR-340, miR-219, miR-338, miR-642, miR-181b, miR-18a,miR-190, miR-213, miR-330, miR-181d, miR-151, miR-140) PreeclampsiamiR-210 (miR-152) Gestational diabetes (miR-29a, miR-132) Plateletactivity miR-126, miR-197, miR-223, miR-24, miR-21 Pregnancy/ pl acenta-miR-526a, miR-527, miR-520d-5p, miR-141, miR-149, miR-299-5p, derivedmiR-517a Drug treatment for miR-130a, miR-146b, miR-143, miR-145,miR-99b, miR-125a, miR- immunomodulation 204, miR-424, miR-503 Aging(miR-151a-3p, miR-181a-5p, miR-1248) *miRNA markers in parentheses aredownregulated

In some embodiments, the devices, systems and methods of the inventioncan be used to detect or analyze an environmental sample. Anenvironmental sample can be obtained from any suitable source, such as ariver, ocean, lake, rain, snow, sewage, sewage processing runoff,agricultural runoff, industrial runoff, water, tap water or drinkingwater, etc.).

In some embodiments, an analyte that can be detected or analyzed usingthe devices, systems and methods of the invention is an environmentalmarker. An environmental marker can be any suitable marker that can becaptured by a capturing agent that specifically binds the environmentalmarker in a device configured with the capturing agent. In someembodiments, the devices, systems and methods of the present inventiondetect the concentration of lead or toxins in water. In someembodiments, the presence, absence, or quantitative level of anenvironmental marker in a sample can be indicative of the state of theenvironment from which the sample was obtained. In some embodiments, theenvironmental marker can be a substance that is toxic or harmful to anorganism, e.g., human, companion animal, plant, etc., that is exposed tothe environment. In some embodiments, the environmental marker can be anallergen that can cause allergic reactions in some individuals who areexposed to the environment. In some embodiments, the presence, absenceor quantitative level of the environmental marker in the sample can becorrelated with a general health of the environment. In such cases, thegeneral health of the environment can be measured over a period of time,for example, such as a week, months, years, or decades.

In some embodiments, the devices, systems and methods of the presentinvention further include receiving or providing a report that indicatesthe safety or harmfulness for a subject to be exposed to the environmentfrom which the sample was obtained based on information including themeasured amount of the environmental marker. The information used toassess the safety risk or health of the environment can include dataother than the type and measured amount of the environmental marker.These other data can include, for example, the location, altitude,temperature, time of day/month/year, pressure, humidity, wind directionand speed, weather, etc. The data can represent, for example, an averagevalue or trend over a certain period (minutes, hours, days, weeks,months, years, etc.) or an instantaneous value over a shorter period(milliseconds, seconds, minutes, etc.).

In some embodiments, the report can be generated by the deviceconfigured to read the device, or can be generated at a remote locationupon sending the data including the measured amount of the environmentalmarker. In some embodiments, an expert can be at the remote location orhave access to the data sent to the remote location, and can analyze orreview the data to generate the report. In some embodiments, the expertcan be a scientist or administrator at a governmental agency, such asthe US Centers for Disease Control (CDC) or the US EnvironmentalProtection Agency (EPA), a research institution, such as a university,or a private company. In some embodiments, the expert can send to theuser instructions or recommendations based on the data transmitted bythe device and/or analyzed at the remote location.

A list of exemplary environmental markers is set forth in Table 8 ofU.S. provisional application Ser. No. 62/234,538, filed on Sep. 29,2015, which application is incorporated by reference herein.

Additional exemplary environmental markers are listed in Table 4.8.

TABLE 4.8 Environmental markers Class/Source Marker Synthetic17beta-estradiol (E2), estrone (El), estrogen (ES: El + E2 + estriol(E3)), 1 hormone 7alfa-ethynylestradiol (EE2), 4-nonylphenpol,testosterone analogues Halogenated p,p′-DDE, p,p′-DDD, p,p′-DDT,o,p′-DDE, o,p′-DDE, o,p′-DDT, o,p′-DDD, hydrocarbons chlordane,nonachlor, oxychlordane, heptachlor, heptachlor epoxide,pentachloroanisole, hexachlorobenzene, heptachlorbenzene, o,p′-methoxychlor, p,p′-methoxychlor, Hexachlorocyclopentadiene Pesticidesmanganese ethylene-bis-dithiocarbamate, diazinon, chlorphyrifos,carbofuran, carbaryl, malathion, dieldrin, fipronil, desulfinylfipronil,fipronil sulfide, fipronil sulfone, aldicarb, aldicarb sulfone, aldicarbsulfoxide, carbaryl, 3- hydroxycarbofuran, methiocarb, methomyl, oxamyl,propoxur, alpha-HCH, gamma-HCH, beta-HCH, delta-HCH, azinphos-methyl,chlorpyrifos, disulfoton, parathion, fonofos, ethoprop,parathion-methyl, phorate, terbufos, cis-permethrin, trans-permethrin,propargite, aldrin, chloroneb, endosulfan I, endrin, isodrin, mirex,toxaphene, lindane, O-ethyl O-4-nitrophenyl phenylphosphono-thioate(EPN), fenitrothion, pirimiphos-methyl, deltamethrin Herbicideacetochlor, alachlor, metolachlor, atrazine, deethylatrazine, cyanazine,terbuthylazine, terbutryn, metribuzin, bentazon, EPTC, triflualin,molinate norflurazon, simazine, prometon, promteryn, tebuthiuron, 2,4-D,diuron, dacthal, bromacil, deisopropyl atrazine, hydroxyatrazine,deethylhydroxyatrazine, deisopropylhydroxy atrazine, acetochlor ESA,acetochlor OA, alachlor ESA, alachlor OA, metolachlor ESA, metolachlorOA, 2,6-diethylaniline, napropamide, pronamide, propachlor, propanilmbutylate, pebulate, propham, thiobencarb, triallate, dacthal, dacthalmonoacid, 2,4-DB, dischlorprop, MCPA, MCPB, 2,4,5-T, 2,4,5-TP,benfluralin, ethalfluralin, oryzalin, pendimethalin, trifluralin,bentazon, norflurazon, acifluorfen, chloramben methyl ester, clopyralid,dicamba, picloram, dinoseb, DNOC, chlorothalonil, dichlobenil,2,6-dichlorobenzamide (BAM), triclopyr, bromoxynil, bromacil, terbacil,fenuron, fluometuron, linuron, neburon, dalapon, diquat, endothall,Glyphosate, N-dealkylated triazines, mecoprop Industrial chromatedcopper arsenate, Carbon tetrachloride, Chlorobenzene, p- material/wasteDichlorobenzene, 1,2-Dichloroethanem, 1,1-Dichloroethylene, cis-1,2-Dichloroethylene, trans-1,2-Dichloroethylene, Dichloromethane, Di(2-ethylhexyl) adipate, Di(2-ethylhexyl) phthalate, Dibutyl phthalate(DBP), diethyl phthalate (DEP), dicyclohexyl phthalate (DCHP), Dioxin(2,3,7,8- TCDD), Epichlorohydrin, Ethylene dibromide, Polychlorinatedbiphenyls, Pentachlorophenol, styrene, Tetrachloroethylene, Toluenediisocyanate (TDI), 1,2,4-Tri chlorobenzene, 1,1,1-Tri chloroethane,1,1,2-Trichloroethane, Trichloroethylene, perchloroethylene, Vinylchloride, Xylenes, alkylphenol (AP), AP + APE, bisphenol A (BPA),benzene, Xylene, Toluene, Styrene, Toluidine, 2-(p-Tolyl)ethylamine,Ethylbenzene, 2-Methyl-naphthalene, and Propyl-benzene, PAH (polynucleararomatic hydrocarbons) Drinking water Bromate, Chlorite, Haloaceticacids, Total Trihalomethanes, Chloramines, Chlorine, Chlorine dioxide,Benzo(a)pyrene, 4-tert-octylphenol Household Acrylamide, linearalkylbenzene sulfonates (LAS), alkyl ethoxylates (AE), waste/ Sewagealkylphenol ethoxylates (APE), triclosan runoff Poison/toxinsN-methylamino-L-alanine (BMAA), Clostridium botulinum neurotoxins, BoNTA, B, D, E, Ricin A, B, tetanus toxin, diphtheria toxin, pertussis toxinHeavy metal mercury/methylmercury, lead/tetraethyl lead, zinc, copper,nickel, cadmium, chromium (VI)/chromate, aluminum, iron, arsenic,cobalt, selenium, silver, antimony, thallium, polonium, radium, tin,metallothionein (in carp liver tissue) Other Lithium, beryllium,manganese, barium, cyanide, fluoride metals/inorganic chemicalsPathogens/ Anthrax (LF), Giardia lamblia, Legionella, Total Coliforms(including fecal microbes (antigen coliform and E. Coli), Viruses(enteric) stapylococci (e.g., Staphylococcus in pretheses) epidermidisand Staphylococcus aureus (enterotoxin A, B, C, G, I, cells, TSST- 1),Enterrococcus faecalis, Pseudomonas aeruginosa, Escherichia coli (Shiga-like toxin, F4, F5, H, K, O, bacteriophage K1, K5, K13), othergram-positive bacteria, and gram-negative bacilli. Clostridium difficile(Toxin A, B) Bacteroidetes, Cryptosporidium parvum (GP900, p68 orcryptopain, oocyst), Candida albicans Bacillus anthracis, Bacillusstearothermophilus Norovirus, Listeria monocytogenes (internalin),Leptospira interrogans, Leptospira biflexa, Clostridium perfringens(Epsilon toxin), Salmonella typhimurium, Yersinia pestis (F1, Vantigens), Aspergillus flavus (aflatoxin), Aspergillus parasiticus(aflatoxin), avian influenza virus, Ebola virus (GP), Histoplasmacapsulatum, Blastomyces dermatitidis (A antigen) Gram-positive bacteria(teichoic acid), Gram-ngative bacteria (such as Pseudomonas aeruginosa,Klebsiella pneumoniae, Salmonella enteriditis, Enterobacter aerogenes,Enterobacter hermanii, Yersinia enterocolitica and Shigellasonnei)(LPS), Polio virus, Influenza type A virus Disease specific prion(PrP-d) Allergens mite (Acas13, Blot1, Blot3, Blot4, Blot5, Blot6,Blot10, Blot11, Blot12, Blot13, Blot19); American house dust mite(Derf1, Derf2, Derf3, Derf7, Derf10, Derf11, Derf14, Derf15, Derf16,Derf17, Derf18w); house dust mite (Derm1); European house dust mite(Derp1, Derp2, Derp3, Derp4, Derp5, Derp6, Derp7, Derp8, Derp9, Derp10,Derp11, Derp14, Derp20, Derp21); mite (Eurm2; Eurm14); storage mite(Glyd2, Lepd2, Lepd5, Lepd7, Lepd10, Lepd13, Tyrp2, Tyrp13);Dermatophagoides farinae (Derf1.0101, Derf1.0102, Derf1.0103,Derf1.0104, Derf1.0105, Derf2.0101, Derf2.0102, Derf2.0103, Derf2.0104,Derf2.0105, Derf2.0106, Derf2.0107, Derf2.0108, Derf2.0109, Derf2.0110,Derf2.0111, Derf2.0112, Derf2.0113, Derf2.0114, Derf2.0115, Derf2.0116,Derf2.0117); Dermatophagoides pteronyssinus (Derp1.0101, Derp1.0102,Derp1.0103, Derp1.0104, Derp1.0105, Derp1.0106, Derp1.0107, Derp1.0108,Derp1.0109, Derp1.0110, Derp1.0111, Derp1.0112, Derp1.0113, Derp1.0114,Derp1.0115, Derp1.0116, Derp1.0117, Derp1.0118, Derp1.0119, Derp1.0120,Derp1.0121, Derp1.0122, Derp1.0123, Derp2.0101, Derp2.0102, Derp2.0103,Derp2.0104, Derp2.0105, Derp2.0106, Derp2.0107, Derp2.0108, Derp2.0109,Derp2.0110, Derp2.0111, Derp2.0112, Derp2.0113); Euroglyphus maynei(Eurm2.0101, Eurm2.0102); Glycyphagus domesticus (Glyd2.0101,Glyd2.0201); and Lepidoglyphus destructor (Lepd2.0101, Lepd2.0101,Lepd2.0101, Lepd2.0102, Lepd2.0201, Lepd2.0202) Pollen (Short Ragweed(Ambrosia artemisiifolia) allergen, Amb a 1, Amba2, Amba3, Amba5, Amba6,Amba7, Amba8, Amba9, Amba10; Betula verrucosa allergen, Bet v 1, Phleumpratense allergen, Phl p 5), giant ragweed (Ambt5); mugwort (Artv1,Artv2, Artv3, Artv4, Artv5, Artv6); sunflower (Hela1, Hela2, Hela3);Mercurialis annua (Mera1); lamb’s-quarters, pigweed (Chea1); whitegoosefoot (Chea2, Chea3); Russian-thistle (Saiki); Rosy periwinkle(Catr1); English plantain (Plal1); Japanese hop (Humj 1); Parietariajudaica (Parj1, Parj2, Parj3); Parietaria officinalis (Paro1); Ambrosiaartemisiifolia (Amba8.0101, Amba8.0102, Amba9.0101, Amba9.0102);Plantago lanceolata (Plal1.0101, Plal1.0102, Plal1.0103); and Parietariajudaica (Parj1.0101, Parj1.0102, Parj1.0201, Par2.0101, Parj2.0102,Parj3.0101, Parj3.0102), Bermuda grass (Cynd1, Cynd7, Cynd12, Cynd15,Cynd22w, Cynd23, Cynd24); orchard grass (Dacg1, Dacg2, Dacg3, Dacg5);meadow fescue (Fesp4w); velvet grass (Holl1); rye grass (Lolp1, Lolp2,Lolp3, Lolp5, Lolpl1); canary grass (Phaa1); Timothy (Phlp1, Phlp2,Phlp4, Phlp5, Phlp6, Phlp11, Phlp12, Phlp13); Kentucky blue grass(Poap1, Poap5); Johnson grass (Sorh1); Cynodon dactylon (Cynd1.0101,Cynd1.0102, Cynd1.0103, Cynd1.0104, Cynd1.0105, Cynd1.0106, Cynd1.0107,Cynd1.0201, Cynd1.0202, Cynd1.0203, Cynd1.0204); Holcus lanatus(Holl1.0101, Holl1.0102); Lolium perenne (Lolp1.0101, Lolp1.0102,Lolp1.0103, Lolp5.0101, Lolp5.0102); Phleum pretense (Phlp1.0101,Phlp1.0102, Phlp4.0101, Phlp4.0201, Phlp5.0101, Phlp5.0102, Phlp5.0103,Phlp5.0104, Phlp5.0105, Phlp5.0106, Phlp5.0107, Phlp5.0108, Phlp5.0201,Phlp5.0202); and Secale cereale (Secc20.0101, Secc20.0201), Alder(Alng1); Birch (Betv1, Betv2, Betv3, Betv4, Betv6, Betv7); hornbeam(Carb1); chestnut (Cass1, Cass5, Cass8); hazel (Cora1, Cora2, Cora8,Cora9, Cora10, Cora11); White oak (Quea1); Ash (Frae1); privet (Ligv1);olive (Olee1, Olee2, Olee3, Olee4, Olee5, Olee6, Olee7, Olee8, Olee9,Olee1O); Lilac (Syrv1); Sugi (Cryj1, Cryj2); cypress (Cupa1); commoncypress (Cups1, Cups3w); mountain cedar (Juna1, Juna2, Juna3); pricklyjuniper (Juno4); mountain cedar (Juns1); eastern red cedar (Junv1);London plane tree (Plaa1, Plaa2, Plaa3); date palm (Phod2); Betulaverrucosa (Betv1.0101, Betv1.0102, Betv1.0103, Betv1.0201, Betv1.0301,Betv1.0401, Betv1.0402, Betv1.0501, Betv1.0601, Betv1.0602, Betv1.0701,Betv1.0801, Betv1.0901, Betv1.1001, Betv1.1101, Betv1.1201, Betv1.1301,Betv1.1401, Betv1.1402, Betv1.1501, Betv1.1502, Betv1.1601, Betv1.1701,Betv1.1801, Betv1.1901, Betv1.2001, Betv1.2101, Betv1.2201, Betv1.2301,Betv1.2401, Betv1.2501, Betv1.2601, Betv1.2701, Betv1.2801, Betv1.2901,Betv1.3001, Betv1.3101, Betv6.0101, Betv6.0102); Carpinus betulus(Carb1.0101, Carb1.0102, Carb1.0103, Carb1.0104, Carb1.0105, Carb1.0106,Carb1.0106, Carb1.0106, Carb1.0106, Carb1.0107, Carb1.0107, Carb 1.0108,Carb 1.0201, Carb 1.0301, Carb 1.03 02); Corylus avellana (Cora1.0101,Cora1.0102, Cora1.0103, Cora1.0104, Cora1.0201, Cora1.0301, Cora1.0401,Cora1.0402, Cora1.0403, Cora1.0404); Ligustrum vulgare (Ligv1.0101,Ligv1.01.02); Olea europea (Olee1.0101, Olee1.0102, Olee1.0103,Olee1.0104, Olee1.0105, Olee1.0106, Olee1.0107); Syringa vulgaris(Syrv1.0101, Syrv1.0102, Syrv1.0103); Cryptomeria japonica (Cryj2.0101,Cryj2.0102); and Cupressus sempervirens (Cups1.0101, Cups1.0102,Cups1.0103, Cups1.0104, Cups1.0105) mold (Alternaria alternata allergen,Alt a 1, Alta3, Alta4, Alta5, Alta6, Alta7, Alta8, Alta10, Alta12,Alta13, Aspergillus fumigatus allergen, Asp f 1, Aspf2, Aspf3, Aspf4,Aspf5, Aspf6, Aspf7, Aspf8, Aspf9, Aspf10, Aspf11, Aspf12, Aspf13,Aspf15, Aspf16, Aspf17, Aspf18, Aspf22w, Aspf23, Aspf27, Aspf28,Aspf29); Aspergillus niger (Aspn14, Aspn18, Aspn25); Aspergillus oryzae(Aspo13, Aspo21); Penicillium brevicompactum (Penb13, Penb26);Penicillium chrysogenum (Pench13, Pench18, Pench20); Penicilliumcitrinum (Penc3, Penc13, Penc19, Penc22w, Penc24); Penicillium oxalicum(Peno18); Fusarium culmorum (Fusc1, Fusc2); Trichophyton rubrum (Trir2,Trir4); Trichophyton tonsurans (Trit1, Trit4); Candida albicans (Canda1,Canda3); Candida boidinii (Candb2); Psilocybe cubensis (Psic1, Psic2);shaggy cap (Cope1, Copc2, Copc3, Copc5, Copc7); Rhodotorula mucilaginosa(Rhom1, Rhom2); Malassezia furfur (Malaf2, Malaf3, Malaf4); Malasseziasympodialis (Malas1, Malas5, Malas6, Malas7, Malas8, Malas9, Malas10,Malas11, Malas12, Malas13); Epicoccum purpurascens (Epip1); andAlternaria alternate (Alta1.0101, Alta1.0102), Aspergillus versicolorantigen, S. chartarum antigen), Cladosporium herbarum (Clah2, Clah5,Clah6, Clah7, Clah8, Clah9, Clah10, Clah12); Aspergillus flavus(Aspf113); animals (Bos domesticus dander allergen, Bos d 2, Bosd3,Bosd4, Bosd5, Bosd6, Bosd7, Bosd8, Bosd2.0101, Bosd2.0102, Bosd2.0103,Canis familiaris allergen, Can f 1,Canf2, Canf3, Canf4, Equus caballusallergen, Equc1, Equc2, Equc3, Equc4, Equc5, Felis domesticus allergen,Fel d 1, Feld2, Feld3, Feld4, Feld5w, Feld6w, Feld7w, guinea pig (Cavp1,Cavp2); Mouse Urinary Protein (MUP, Musm1) allergen, Mus m 1, RatUrinary Protein (RUP, Ratn1) allergen, Rat n 1., Equus caballus(Equc2.0101, Equc2.0102)) Mosquito (Aeda1, Aeda2); honey bee (Apim1,Apim2, Apim4, Apim6, Apim7); bumble bee (Bomp1, Bomp4); German cockroach(Blag1, Blag2, Blag4, Blag5, Blag6, Blag7, Blag8); American cockroach(Pera1, Pera3, Pera6, Pera7); midge (Chit1-9, Chit1.01, Chit1.02,Chit2.0101, Chit2.0102, Chit3, Chit4, Chit5, Chit6.01, Chit6.02, Chit7,Chit8, Chit9); cat flea (Ctef1, Ctef2, Ctef3); pine processionary moth(Thap1); silverfish (Leps1); white face hornet (Dolm1, Dolm2, Dolm5);yellow hornet (Dola5); wasp (Pola1, Pola2, Pola5, Pole1, Pole5, Polf5,Polg5, Polm5, Vesvi5); Mediterranean paper wasp (Pold1, Pold4, Pold5);European hornet (Vespc1, Vespc5); giant asian hornet (Vespm1, Vespm5);yellowjacket (Vesf5, Vesg5, Vesm1, Vesm2, Vesm5, Vesp5, Vess5, Vesv1,Vesv2, Vesv5); Australian jumper ant (Myrp1, Myrp2); tropical fire ant(Solg2, Solg4); fire ant (Soli2, Soli3, Soli4); Brazilian fire ant(Sols2); California kissing bug (Triap1); Blattella germanica(Blag1.0101, Blag1.0102, Blag1.0103, Blag1.02, Blag6.0101, Blag6.0201,Blag6.0301); Periplaneta Americana (Pera1.0101, Pera1.0102, Pera1.0103,Pera1.0104, Pera 1.02, Pera3.01, Pera3.0201, Pera3.0202, Pera3.0203,Pera7.0101, Pera7.0102); Vespa crabo (Vespc5.0101, Vespc5.0101); andVespa mandarina (Vesp m 1.01, Vesp m 1.02) Nematode (Anis1, Anis2,Anis3, Anis4); pigeon tick (Argr1); worm (Ascs1); papaya (Carp1); softcoral (Denn1); rubber (latex)(Hevb1, Hevb2, Hevb3, Hevb4, Hevb5,Hevb6.01, Hevb6.02, Hevb6.03, Hevb7.01, Hevb7.02, Hevb8, Hevb9, Hevb10,Hevb11, Hevb12, Hevb13); human autoallergens (Homs1, Homs2, Homs3,Homs4, Homs5); obeche (Trips1); and Hevea brasiliensis (Hevb6.01,Hevb6.0201, Hevb6.0202, Hevb6.03, Hevb8.0101, Hevb8.0102, Hevb8.0201,Hevb8.0202, Hevb8.0203, Hevb8.0204, Hevb10.0101, Hevb10.0102,Hevb10.0103, Hevb11.0101, Hevb11.0102)

In some embodiments, the devices, systems and methods of the presentinvention can be used to detect or analyze a foodstuff sample. Afoodstuff sample can be obtained from any suitable source, such as fromraw food, processed food, cooked food, drinking water, etc. In someembodiments, an analyte that can be detected or analyzed using thedevices, systems and methods of the invention is an foodstuff marker. Afoodstuff marker can be any suitable marker, such as those shown inTable 4.9, that can be captured by a capturing agent that specificallybinds the foodstuff marker in a device configured with the capturingagent. In some embodiments, the presence, absence, or the quantitativelevel of the foodstuff marker in the sample can be indicative of thesafety or harmfulness to a subject if the foodstuff is consumed. In someembodiments, the foodstuff marker is a substance derived from apathogenic or microbial organism that is indicative of the presence ofthe organism in the foodstuff from which the sample was obtained. Insome embodiments, the foodstuff marker is a toxic or harmful substanceif consumed by a subject. In some embodiments, the foodstuff marker is abioactive compound that can unintentionally or unexpectedly alter thephysiology if consumed by the subject.

In some embodiments, the foodstuff marker is indicative of the manner inwhich the foodstuff was obtained (for example, grown, procured, caught,harvested, processed, cooked, etc.). In some embodiments, the foodstuffmarker is indicative of the nutritional content of the foodstuff. Insome embodiments, the foodstuff marker is an allergen that can induce anallergic reaction if the foodstuff from which the sample is obtained isconsumed by a subject.

In some embodiments, the devices, systems and methods in the presentinvention further includes receiving or providing a report thatindicates the safety or harmfulness for a subject to consume thefoodstuff from which the sample was obtained based on informationincluding the measured level of the foodstuff marker. The informationused to assess the safety of the foodstuff for consumption can includedata other than the type and measured amount of the foodstuff marker.These other data can include any health condition associated with theconsumer (allergies, pregnancy, chronic or acute diseases, currentprescription medications, etc.).

The report can be generated by the device configured to read the device,or can be generated at a remote location upon sending the data includingthe measured amount of the foodstuff marker. In some embodiments, a foodsafety expert can be at the remote location or have access to the datasent to the remote location, and can analyze or review the data togenerate the report. In some embodiments, the food safety expert can bea scientist or administrator at a governmental agency, such as the USFood and Drug Administration (FDA) or the CDC, a research institution,such as a university, or a private company. In some embodiments, thefood safety expert can send to the user instructions or recommendationsbased on the data transmitted by the device and/or analyzed at theremote location.

A list of exemplary foodstuff markers is set forth in Table 9 of U.S.provisional application Ser. No. 62/234,538, filed on Sep. 29, 2015,which application is incorporated by reference herein.

Additional exemplary foodstuff markers are listed in Table 4.9.

TABLE 4.9 Foodstuff markers Source/Class Marker/target Pathogens/micBacillus anthracis (LF), Giardia lamblia, Legionella, Total Coliformsrobes (including fecal coliform and E. Coli), Viruses (enteric)stapylococci (e.g., Staphylococcus epidermidis and Staphylococcus aureus(enterotoxin A, B, C, G, 1, cells, TSST-1), Enterrococcus faecalis,Pseudomonas aeruginosa, Escherichia coli (Shiga-like toxin, F4, F5, H,K, O, bacteriophage K1, K5, K13), other gram-positive bacteria, andgram-negative bacilli. Clostridium difficile (Toxin A, B),Bacteroidetes, Cryptosporidium parvum (GP900, p68 or cryptopain,oocyst), Candida albicans, Bacillus anthracis, Bacillusstearothermophilus, Bacillus cereus, Bacillus licheniformis, Bacillussubtilis, Bacillus pumilus, Bacillus badius, Bacillus globigii,Salmonella typhimurium, Escherichia coli 0157:H7, Norovirus, Listeriamonocytogenes (internalin), Leptospira interrogans, Leptospira biflexa,Campylobacter jejuni, Campylobacter coli, Clostridium perfringens,Aspergillus flavus (aflatoxins), Aspergillus parasiticus (aflatoxins),Ebola virus (GP), Histoplasma capsulatum, Blastomyces dermatitidis (Aantigen), Gram-positive bacteria (teichoic acid), Gram-ngative bacteria(such as Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonellaenteriditis, Enterobacter aerogenes, Enterobacter hermanii, Yersiniaenterocolitica and Shigella sonnei)(LPE), Polio virus, Influenza type Avirus, Disease specific prion (PrP-d), Hepatitis A virus, Toxoplasmagondii, Vibrio cholera, Vibrio parahaemolyticus, Vibrio vulnificus,Enterococcus faecalis, Enterococcus faecium Toxins/carcinoN-methylamino-L-alanine (BMAA), Clostridium botulinum neurotoxins, gensBoNT A, B, Ricin A, B; diphtheria toxin; Aristolochic acid; Colchicine,Ochratoxin A, Sterigmatocystin, Ergotamine, Fumonisins, Fusarin C,domoic acid, Brevetoxin, Mycotoxins Halogenated Heptachlor, chlordanehydrocarbons Heavy metals Lead, mercury, cadmium Allergens peanut (Ara h1, Ara h 2, Ara h 6), fish, shellfish, mollusks, shrimp (D.pteronyssinus tropomyosin allergen, Der p 10) Cod (Gadc1); Atlanticsalmon (Sals1); domestic cattle milk (Bosd4, Bosd5, Bosd6, Bosd7,Bosd8); chicken/egg (Gald1, Gald2, Gald3, Gald4, Gald5); shrimp (Mete1);shrimp (Pena1, Peni1); black tiger shrimp (Penm1, Penm2); squid (Todp1),brown garden snail (Helas1); abalone (Halm1); edible frog (Rane1,Rane2); oriental mustard (Braj1); rapeseed (Bran1); cabbage (Brao3);turnip (Brar1, Brar2); barley (Horv15, Horv16, Horv17, Horv21); rye(Secc20); wheat (Tria18, Tria19, Tria25, Tria26, gliadin); corn (Zeam14,Zeam25); rice (Orys1), celery (Apig1, Apig4, Apig5); carrot (Dauc1,Dauc4); hazelnut (Cora1.04, Cora2, Cora8); strawberry (Fraa1, Fraa3,Fraa4); apple (Mald1, Mald2, Mald3, Mald4); pear (Pyrc1, Pyrc4, Pyrc5);avocado (Persa1); apricot (Pruar1, Pruar3); sweet cherry (Pruav1,Pruav2, Pruav3, Pruav4); European plum (Prud3); almond (Prudu4); peach(Prup3, Prup4); asparagus (Aspao1); saffron crocus (Cros1, Cros2);lettuce (Lacs1); grape (Vitv1); banana (Musxp1); pineapple (Anac1,Anac2); lemon (Citl3); sweet orange (Cits1, Cits2, Cits3); litchi(Litc1); yellow mustard (Sinai); soybean (Glym1, Glym2, Glym3, Glym4);mung bean (Vigr1); peanut (Arah1, Arah2, Arah3, Arah4, Arah5, Arah6,Arah7, Arah8); lentil (Lenc1, Lenc2); pea (Piss1, Piss2); kiwi (Actc1,Actc2); bell pepper (Capa1w, Capa2); tomato (Lyce1, Lyce2, Lyce3);potato (Solat1, Solat2, Solat3, Solat4); Brazil nut (Bere1, Bere2);black walnut (Jugn1, Jugn2); English walnut (Jugr1, Jugr2, Jugr3);Cashew (Anao1, Anao2, Anao3); Castor bean (Ricc1); sesame (Sesi1, Sesi2,Sesi3, Sesi4, Sesi5, Sesi6); muskmelon (Cucm1, Cucm2, Cucm3);Chinese-date (Zizm1); Anacardium occidentals (Anao1.0101, Anao 1.0102);Apium graveolens (Apig1.0101, Apig1.0201); Daucus carota (Dauc1.0101,Dauc1.0102, Dauc1.0103, Dauc1.0104, Dauc1.0105, Dauc1.0201); Citrussinensis (Cits3.0101, Cits3.0102); Glycine max (Glym1.0101, Glym1.0102,Glym3.0101, Glym3.0102); Lens culinaris (Lend.0101, Lend.0102,Lend.0103); Pisum sativum (Piss1.0101, Piss1.0102); Lycopersiconesculentum (Lyce2.0101, Lyce2.0102); Fragaria ananassa (Fraa3.0101,Fraa3.0102, Fraa3.0201, Fraa3.0202, Fraa3.0203, Fraa3.0204, Fraa3.0301);Malus domestica (Mald1.0101, Mald1.0102, Mald1.0103, Mald1.0104,Mald1.0105, Mald1.0106, Mald1.0107, Mald1.0108, Mald1.0109, Mald1.0201,Mald1.0202, Mald1.0203, Mald1.0204, Mald1.0205, Mald1.0206, Mald1.0207,Mald1.0208, Mald1.0301, Mald1.0302, Mald1.0303, Mald1.0304, Mald1.0401,Mald1.0402, Mald1.0403, Mald3.0101w, Mald3.0102w, Mald3.0201w,Mald3.0202w, Mald3.0203w, Mald4.0101, Mald4.0102, Mald4.0201,Mald4.0202, Mald4.0301, Mald4.0302); Prunus avium (Pruav1.0101,Pruav1.0201, Pruav1.0202, Pruav1.0203); and Prunus persica (Prup4.0101,Prup4.0201) Synthetic 17beta-estradiol (E2), estrone (E1), estrogen (ES:El + E2 + estradiol (E3)), hormone 1 7alfa-ethynylestradiol (EE2),4-nonylphenpol, testosterone, analogues Diethylstilbestrol (DES),recombinant bovine growth hormone (rBGH) Pesticides Dieldrin, carbaryl,chlorpyrifos, parathion, aldrin, endosulfan I, endrin, toxaphene,O-ethyl O-4-nitrophenyl phenylphosphono-thioate (EPN), fenitrothion,pirimiphos-methyl, thiabendazole, methiocarb, Carbendazim, deltamethrin,Avermectin, Carbaryl, Cyanazine, Kresoxim, resmethrin, kadethrin,cyhalothrin, biphenthrin, fenpropathrin, allethrin and tralomethrin;aromatic-substituted alkanecarboxylic acid esters such as fenvarerate,flucythrinate, fluvalinate and cycloprothrin; and non-ester compoundssuch as etofenprox, halfenprox (MTI-732), 1-(3-phenoxyphenyl)-4-(4-ethoxyphenyl)-4-methylpentane (MTI-790), 1-(3-phenoxy-4-fluorophenyl)-4-(4-ethoxyphenyl)-4-methylpentane (MTI-800),dimethyl-(4-ethoxyphenyl)-(3-phenoxybenzyloxy)silane (SSI-116),silafluofen and PP-682, carbofuran, triazophos Herbicide atrazine,deethylatrazine, cyanazine, terbuthylazine, terbutryn, molinate,simazine, prometon, promteryn, hydroxyatrazine, 2,6-dichlorobenzamide(BAM), N-dealkylated triazines, mecoprop, thiram, acetochlor, alachlor,Chlorothalonil, Chlorsulfuron, Fenoxaprop ethyl, Linuron, monuron,diuron, Quizalofop-ethyl, Imazalil, Iprodione, Iprovalicarb,Myclobutanil Industrial Dioxin (2,3,7,8-TCDD), 4-tert-octylphenol,bisphenol A (BPA), Styrene, materi al/waste Di(2-ethylhexyl) phthalate,Dibutyl phthalate (DBP), benzophenone, benzene, trichloroethylene,polychlorinated biphenyl (PCB), nonylphenol, p-cresol, melamine, xyleneAntibiotics 3-Amino-5-morpholinomethyl-2-oxazolidone (AMOZ; tissue boundmetabolite of furaltadone), oxytetracycline, rolitetracycline,Actinomycin D, Amikacin sulfate, Aminoglycosides, nitrofuran (AOZ),Chloramphenicol, Doxycycline, Streptomycin, gentamicin, neomycin,kanamycin, sulfamethazine, enrofloxacin, sulfadiazine, enrofloxacin Foodcoloring Tartrazine, ethoxyquin, erythritol, penicillin,Fluoroquinolone, Malachite /additive/ Green/Leucomalachite Green, C.I.Solvent Yellow 14 (Sudan I), preservative Food Acrylamide,2-amino-3-methylimidazo(4,5-f)quinolone, Benzo[a]pyrene preparationNutritional Vitamins A (retinol), B12 (cobalmins), B6 (pyridoxine), B1(thiamin), B2 content (riboflavin), B3 (niacin), B5 (D-pantothenicacid), B7 (biotin), B9 (folic acid), C, D, E (alpha-tocopherol); OtherCaffeine, Ovine myofibril proteins, Etodolac

In some embodiments, the invention is directed to a kit containing adevice of the invention. In some embodiments, the kit includes a deviceconfigured to specifically bind an analyte described herein. In someembodiments, the kit includes instructions for practicing the subjectmethods using a hand held device, e.g., a mobile phone. In someembodiments, the instructions can be present in the kits in a variety offorms, one or more of which can be present in the kit. One form in whichthese instructions can be present is as printed information on asuitable medium or substrate, e.g., a piece or pieces of paper on whichthe information is printed, in the packaging of the kit, in a packageinsert, etc. Another means would be a computer readable medium, e.g.,diskette, CD, DVD, Blu-Ray, computer-readable memory, etc., on which theinformation has been recorded or stored. Yet another means that can bepresent is a website address which can be used via the Internet toaccess the information at a removed site. The kit can further include asoftware for implementing a method for measuring an analyte on a device,as described herein, provided on a computer readable medium. Anyconvenient means can be present in the kits.

In some embodiments, the kit includes a detection agent that includes adetectable label, e.g. a fluorescently labeled antibody oroligonucleotide that binds specifically to an analyte of interest, foruse in labeling the analyte of interest. The detection agent can beprovided in a separate container as the device, or can be provided inthe device.

In some embodiments, the kit includes a control sample that includes aknown detectable amount of an analyte that is to be detected in thesample. The control sample can be provided in a container, and can be insolution at a known concentration, or can be provided in dry form, e.g.,lyophilized or freeze dried. The kit can also include buffers for use indissolving the control sample, if it is provided in dry form.

In some embodiments, the devices, systems and methods of the inventioncan be used for simple, rapid blood cell counting using a smartphone. Insome embodiments, the first plate and the second plate are selected froma thin glass slide (e.g., 0.2 mm thick) or a thin plastic film (e.g., 15mm thick) of a relative flat surface, and each have an areas with alength and width in about 0.5 cm to 10 cm. In some embodiments, thespacers are made of glass, plastics, or other materials that would notdeform significantly under a pressing. In some embodiments, before thesample deposition, the spacers are placed on the first plate, the secondplate or both; and the first plate, the second plate or both areoptionally coated with reagent that facilitate the blood counting(staining dyes and/or anticoagulant). In some embodiments, the firstplate and the second plate can be sealed in a bag for easy transport andlonger shelf life-time.

In some embodiments of blood cell count testing, only about 1 uL(microliter) (or about 0.1 uL to 3 uL) of blood is needed for thesample, which can be taken, for example, from a finger or other humanbody location. In some embodiments, the blood sample can be directlydeposited from human body (e.g., finger) onto the first plate and thesecond plate, without any dilution. In such embodiments, the first plateand the second plate can face each other, so that blood sample isbetween the inner surfaces of the first plate and the second plate. Insome embodiments, reagents are pre-deposited (staining dyes oranticoagulant), they are deposited on the inner surface for mixing withthe sample. The first plate and the second plate can then be pressed byfingers or a simple mechanical device (e.g. a clip that presses using aspring). Under the press, the inner spacing is reduced, the reductionwill be eventually stopped at the value set by the spacers' height andthe final sample thickness is reached, which generally is equal to thefinal inner spacing. Since the final inner spacing is known, the finalsample thickness become known, namely being quantified (measured) bythis method.

In some embodiments, if the blood sample is not diluted, after pressing(sample deformation) the spacers and hence the final sample thicknesscan be thin, e.g., less than 1 um, less than 2 um, less than 3 um, lessthan 4 um, less than 5 um, less than 7 um, less than 10 um, less than 15um, less than 20 um, less than 30 um, less than 40 um, less than 50 um,less than 60 um, less than 80 um, less than 100 um, less than 150 um, orany ranges between any of the two numbers. A thin final sample can beuseful because if the final sample thickness is thick, then many redcells can overlap during the imaging, which can make the cell countinginaccurate. For example, about 4 um thick of whole blood withoutdilution will give about one layer of blood red cells.

After the pressing, the sample can be imaged by a smartphone eitherdirectly or through an additional optical elements (e.g. lenses,filters, or light sources as needed). The image of the sample can beprocessed to identify the types of the cells as well as the cell number.The image processing can be done locally at the same smartphone thattakes the image or remotely but the final result transmitted back to thesmartphone (where the image is transmitted to a remote location and isprocessed there.) The smart phone will display the cell number for aparticular cell. In some cases, certain advices will be displayed. Theadvices can stored on the smartphone before the test or come from aremote machines or professionals.

In certain embodiments, reagents are placed on the inner surfaces of thefirst plate and/or the second plate using the methods and devicesdescribed herein.

In some embodiments, a device or a method for the blood testingcomprises (a) a device or a method described herein and (b) a platespacing (i.e. the distance between the inner surfaces of the two plates)at the closed configuration or a use of such spacing, wherein aundiluted whole blood in the plate-spacing has an average inter-celldistance in the lateral direction for the red blood cells (RBC) largerthan the average diameter of the disk shape of the RBC.

In some embodiments, a device or a method to arrange the orientation ofa non-spherical cell comprises (a) a device or a method in as describedherein and (b) a plate spacing (i.e. the distance between the innersurfaces of the two plates) at the closed configuration or a use of suchspacing, wherein the spacing less than the average size of the cell inits long direction (the long direction is the maximum dimensiondirection of a cell). Such arrangement can improve the measurements ofthe sample volume (e.g. red blood cell volume).

In some embodiments, the analytes in the blood tests include proteinmarkers, a list of which can be found at the website of the AmericanAssociation for Clinical Chemistry).

Table 4.10 provides additional exemplary analytes that can be detectedusing the present invention at the point-of-care (POC) settings and/orat the point of use by non-professional users/subjects.

TABLE 4.10 POC analytes Disease/ Condition Analyte 1. HaematologyComplete blood RBCs, WBCs, Platelets count (CBC) 2. Lipid panelCholesterol level Triglyceride, Total cholesterol, HDL cholesterol, LDLcholesterol 3. Urinalysis Renal Diseases/ pH, Protein, Glucose,Nitrites, Leukocyte esterase, Ketones, Blood cells, Kidney FunctionCasts, Crystals, Microorganisms, Squamous cells 4. Diabetes DiabetesGlucose, HbA1c, 11-8, CTSS, ITGB2, HLA-DRA, CD53, PLAG27, or MMP9; RBP4;8-iso-prostaglandin F2a (8-iso-PGF2α), 11-dehydro- thromboxane B2 (TXM),C-peptide, Advanced glycosylation end products (AGEs),1,5-anhydroglucitol, NGPTL3 and 4, autoantibodies (Zn transporter 8,glutamic acid decarboxylase (GAD)), ATP-binding cassette, sub-family C(CFTR/MRP), member 8; ATP-binding cassette, sub-family C (CFTR/MRP),member 9; angiotensin I converting enzyme (peptidyl- dipeptidase A) 1;adenylate cyclase activating polypeptide 1 (pituitary); adiponectin, C1Qand collagen domain containing; adiponectin receptor 1; adiponectinreceptor 2; adrenomedullin; adrenergic, beta-2-, receptor, surface;advanced glycosylation end product-specific receptor; agouti relatedprotein homolog (mouse); angiotensinogen (serpin peptidase inhibitor,clade A, members); angiotensin II receptor, type 1; angiotensin IIreceptor-associated protein; alpha-2-HS-glycoprotein; v-akt murinethymoma viral oncogene homolog 1; v-akt murine thymoma viral oncogenehomolog 2; albumin; Alstrom syndrome 1; archidonate 12- lipoxygenase;ankyrin repeat domain 23; apelin, AGTRL 1 Ligand; apolipoprotein A-I;apolipoprotein A-II; apolipoprotein B (including Ag(x) antigen);apolipoprotein E; aryl hydrocarbon receptor nuclear translocator; Arylhydrocarbon receptor nuclear translocator-like; arrestin, beta 1;arginine vasopressin (neurophysin 11, antidiuretic hormone, Diabetesinsipidus, neurohypophyseal); bombesin receptor subtype 3; betacellulin;benzodiazepine receptor (peripheral); complement component 3; complementcomponent 4A (Rodgers blood group); complement component 4B (Childoblood group); complement component 5; Calpain- 10; cholecystokinin;cholecystokinin (CCK)-A receptor; chemokine (C-C motif) ligand 2; CD14molecule; CD163 molecule; CD36 molecule (thrombospondin receptor); CD38molecule; CD3d molecule, delta (CD3- TCR complex); CD3g molecule, gamma(CD3-TCR complex); CD40 molecule, TNF receptor superfamily member 5;CD40 ligand (TNF superfamily, members, hyper-IgM syndrome); CD68molecule; cyclin- dependent kinase 5; complement factor D (adipsin);CASP8 and FADD- like apoptosis regulator; Clock homolog (mouse); chymase1, mast cell; cannabinoid receptor 1 (brain); cannabinoid receptor 2(macrophage); cortistatin; carnitine palmitoyltransferase I; carnitinepalmitoyltransferase II; complement component (3b/4b) receptor 1;complement component (3d/Epstein Barr virus) receptor 2; CREB bindingprotein (Rubinstein- Taybi syndrome); C-reactive protein,pentraxin-related; CREB regulated transcription coactivator 2; colonystimulating factor 1 (macrophage); cathepsin B; cathepsin L; cytochromeP450, family 19, subfamily A, polypeptide 1; Dio-2, deathinducer-obliterator 1; dipeptidyl-peptidase 4 (CD26, adenosine deaminasecomplexing protein 2); epidermal growth factor (beta-urogastrone); earlygrowth response 1; epididymal sperm binding protein 1; ectonucleotide;pyrophosphatase/phosphodiesterase 1; E1A binding protein p300;coagulation factorXIII, A1 polypeptide; coagulation factor VIII,procoagulant component (hemophilia A); fatty acid binding protein 4,adipocyte; Fas (TNF receptor superfamily, member 6); Fas ligand (TNFsuperfamily, member 6); free fatty acid receptor 1; fibrinogen alphachain; forkhead box A2; forkhead box O1 A; ferritin; glutamatedecarboxylase 2; galanin; gastrin; glucagon; glucokinase;gamma-glutamyltransferase 1; growth hormone 1; ghrelin/obestatinpreprohormone; gastric inhibitory polypeptide; gastric inhibitorypolypeptide receptor; glucagon-like peptide 1 receptor; guaninenucleotide binding protein (G protein), beta polypeptide 3;glutamic-pyruvate transaminase (alanine aminotransferase); gastrinreleasing peptide (bombesin); gelsolin (amyloidosis, Finnish type);hemoglobin; hemoglobin, beta; hypocretin (orexin); neuropeptide;precursor; hepatocyte growth factor (hepapoietin A; scatter factor);hepatocyte nuclear factor 4, alpha; haptoglobin; hydroxysteroid(11-beta); dehydrogenase 1; heat shock 70 kDa protein 1B; islet amyloidpolypeptide; intercellular adhesion molecule 1 (CD54), human rhinovirusreceptor; interferon, gamma; insulin-like growth factor 1 (somatomedinC); insulin-like growth factor 2 (somatomedin A); insulin-like growthfactor binding protein 1; insulin-like growth factor binding protein 3;inhibitor of kappa light polypeptide gene enhancer in B-cells, kinasebeta; interleukin 10; interleukin 18 (interferon-gamma-inducing factor);interleukin 1, alpha; interleukin 1, beta; interleukin 1 receptorantagonist; interleukin 2; interleukin 6 (interferon, beta 2);interleukin 6 receptor; interleukin 8; inhibin, beta A (activin A,activin AB alpha polypeptide); insulin; insulin receptor; insulinpromoter factor-1; insulin receptor substrate 1; insulin receptorsubstrate-2; potassium inwardly-rectifying channel, subfamily J, member11; potassium inwardly-rectifying channel, subfamily J, member 8;klotho; kallikrein B, plasma (Fletcher factor) 1; leptin (obesityhomolog, mouse); leptin receptor; legumain; lipoprotein, Lp(a);lipoprotein lipase; v- maf musculoaponeurotic brosarcoma oncogenehomolog A (avian); mitogen-activated protein kinase 8; interactingprotein 1; mannose-binding lectin (protein C) 2, soluble (opsonicdefect); melanocortin 4 receptor; melanin-concentrating hormone receptor1; matrix metallopeptidase 12 (macrophage elastase); matrixmetallopeptidase 14 (membrane-inserted); matrix metallopeptidase 2(gelatinase A, 72 kDa gelatinase, 72 kDa type IV collagenase); matrixmetallopeptidase 9 (gelatinase B, 92 kDa gelatinase, 92 kDa type IVcollagenase); nuclear receptor co-repressor 1; neurogenicdifferentiation 1; nuclear factor of kappa light polypeptide geneenhancer in B-cells 1(p105); nerve growth factor, beta polypeptide; non-insulin-dependent Diabetes Mellitus (common, type 2) 1; non-insulin-dependent Diabetes Mellitus (common, type 2) 2; Noninsulin-dependentDiabetes Mellitus 3; nischarin (imidazoline receptor); NF-kappaBrepressing factor; neuronatin; nitric oxide synthase 2A; Niemann-Pickdisease, type C2; natriuretic peptide precursor B; nuclear receptorsubfamily 1, group D, member 1; nuclear respiratory factor 1; oxytocin,prepro-(neurophysin I); purinergic receptor P2Y, G-protein coupled, 10;purinergic receptor P2Y, G-protein coupled, 12; purinergic receptor P2Y,G-protein coupled, 2; progestagen-associated endometrial; protein(placental protein 14, pregnancy-associated endometrialalpha-2-globulin, alpha uterine protein); paired box gene 4; pre-B-cellcolony enhancing factor 1; phosphoenolpyruvate carboxykinase 1 (PEPCK1);proprotein convertase; subtilisin/kexin type 1; placental growth factor,vascular; endothelial growth factor-related protein;phosphoinositide-3-kinase, catalytic, alpha polypeptide;phosphoinositide-3-kinase, regulatory subunit 1 (p85 alpha);phospholipase A2, group XIIA; phospholipase A2, group IID; plasminogenactivator, tissue; patatin-like phospholipase domain containing 2;proopiomelanocortin (adrenocorticotropin/beta-lipotropin/alpha-melanocyte stimulating hormone/beta- melanocytestimulating hormone/beta-endorphin); paraoxonase 1 ESA, PON,Paraoxonase; peroxisome proliferative activated receptor, alpha;peroxisome proliferative activated receptor, delta; peroxisomeproliferative activated receptor, gamma; peroxisome proliferativeactivated receptor, gamma, coactivator 1; protein phosphatase 1,regulatory (inhibitor) subunit 3A (glycogen and sarcoplasmic reticulumbinding subunit, skeletal muscle); protein phosphatase 2A, regulatorysubunit B'(PR 53); protein kinase, AMP-activated, beta 1 non-catalyticsubunit; protein kinase, cAMP-dependent, catalytic, alpha; proteinkinase C, epsilon; proteasome (prosome, macropain) 26S subunit,non-ATPase, 9 (Bridge-1); prostaglandin E synthase;prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase andcyclooxygenase); protein tyrosine phosphatase, mitochondrial 1; PeptideYY retinol binding protein 4, plasma (RBP4); regenerating islet-derived1 alpha (pancreatic stone protein, pancreatic thread protein); resistin;ribosomal protein S6 kinase, 90 kDa, polypeptide 1; Ras-relatedassociated with Diabetes; serum amyloid A1; selectin E (endothelialadhesion molecule 1); serpin peptidase inhibitor, clade A (alpha-1antiproteinase, antitrypsin), member 6; serpin peptidase inhibitor,clade E (nexin, plasminogen activator inhibitor type 1), member 1;serum/glucocorticoid regulated kinase; sex hormone-binding globulin;thioredoxin interacting protein; solute carrier family 2, member 10;solute carrier family 2, member 2; solute carrier family 2, member 4;solute carrier family 7 (cationic amino acid transporter, y+ system),member 1(ERR); SNF1-like kinase 2; suppressor of cytokine signaling 3;v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (avian);sterol regulatory element binding transcription factor 1; solute carrierfamily 2, member 4; somatostatin receptor 2; somatostatin receptor 5;transcription factor 1, hepatic; LF-B1, hepatic nuclear factor (HNF1);transcription factor 2, hepatic, LF-B3, variant hepatic nuclear factor;transcription factor 7-like 2 (T-cell specific, HMG- box); transforminggrowth factor, beta 1 (Camurati-Engelmann disease); transglutaminase 2(C polypeptide, protein-glutamine-gamma- glutamyltransferase);thrombospondin 1; thrombospondin, type I, domain containing 1; tumornecrosis factor (TNF superfamily, member 2); tumor necrosis factor (TNFsuperfamily, member 2); tumor necrosis factor receptor superfamily,member 1A; tumor necrosis factor receptor superfamily, member 1B;tryptophan hydroxylase 2; thyrotropin-releasing hormone; transientreceptor potential cation channel, subfamily V, member 1; thioredoxininteracting protein; thioredoxin reductase 2; urocortin 3 (stresscopin);uncoupling protein 2 (mitochondrial, proton carrier); upstreamtranscription factor 1; urotensin 2; vascular cell adhesion molecule 1;vascular endothelial growth factor; vimentin; vasoactive intestinalpeptide; vasoactive intestinal peptide receptor 1; vasoactive intestinalpeptide receptor 2; von Willebrand factor; Wolfram syndrome 1(wolframin); X-ray repair complementing defective repair in Chinesehamster cells 6; c-peptide; cortisol; vitamin D3; estrogen; estradiol;digitalis-like factor; oxyntomodulin; dehydroepiandrosterone sulfate(DHEAS); serotonin (5-hydroxytryptamine); anti-CD38 autoantibodies;gad65 autoantibody; Angiogenin, ribonuclease, RNase A family, 5;Hemoglobin A1c; Intercellular adhesion molecule 3 (CD50); interleukin 6signal transducer (gp130, oncostatin M receptor); selectin P (granuleembrane protein 140 kDa, antigen CD62); TIMP metallopeptidase inhibitor;Proinsulin; endoglin; interleukin 2 receptor, beta; insulin-like growthfactor binding protein 2; insulin-like growth factor 1 receptor;fructosamine, N-acetyl-beta-d- glucosaminidase, pentosidine, advancedglycation end product, beta2- microglobulin, pyrraline 5. SexuallyTransmitted Diseases Chlamydia bacteria Chlamydia trachomatis Gonorrheabacteria Neisseria gonorrhoeae Syphilis Antibodies, bacterial DNATrichomonas protzoan Trichomoniasis Human DNA or RNA of HPV viruspapillomavirus (HPV) Genital herpes Antibodies Human HIV antigen p24,Antibodies Immunodeficienc y Virus (HIV) 6. Other Infectious DiseasesEbola Antigen, IgM and IgG antibodies, RNA Malaria Antigen, Nucleicacids, Antibodies Hepatitis B and Viral proteins, Antibodies, Viral DNAHepatitis C Influenza Viral proteins, Antibodies, Viral DNA 7. Cardiactesting Cardiac markers Troponin (I orT), Creatine Kinase (CK) andCK-MB, Myoglobin, hs-CRP, BNP and NT-proBNP 8. Female Reproductiontesting Pregnancy test HCG (human chorionic gonadotropin) Ovulation testLH (luteinizing hormone) 9. Drugs of Abuse Alcohol Ethanol, ethylglucuronide Cocaine Cocaine, Benzolecgonine, Ecgonine, Ecgonine MethylEster Heroine Heroine, 6MAM, Morphine PCP PCP, PhencyclidineThienylcyclohexylpiperidine (TCP) Amphetamines Amphetamines (such asD-Amphetamine, D-Methamphetamine, L- Amphetamine, L-Methamphetamine,3,4-Methylenedioxy- methamphetamine (MDMA),3,4-Methylenedioxyamphetamine (MDA), 3,4-Methylenedioxyethylamphetamine(MDEA), Paramethoxyamphetamine (PMA)) Methamphetami D-Methamphetamine,D-Amphetamine, L-Methamphetamine, ne Chloroquine, (+/−) Ephedrine,3,4-Methylenedioxy-methamphetamine (MDMA), 3,4-Methylenedioxyamphetamine(MDA), 3,4- Methylenedioxyethylamphetamine (MDEA), Procaine MDMA(Ecstasy) MDMA, MDA, MDEA, D-Amphetamine, D-Methamphetamine,Paramethoxyamphetamine (PMA) Barbiturates Secobarbital, Phenobarbital,Butalbital, Allobarbital, Alphenal, Amorbarbital, Aprobarbital,Hexobarbital, Butabarbital, Pentobarbital Phenobarbital Phenobarbital,Butalbital, Amobarbital, Secobarbital Benzodiazepines Oxazepam,Alprazolam, Bromazepam, Chlordiazepoxide, Clobazam, Clonazepam,Clorazepate, Delorazepam, Desalkyflurazepam, Diazepam, Estazolam,Fentanyl, Flunitrazepam (Rohypnol ®), Flurazepam, a- Hydroxyalprazolam,Lorazepam (Ativan ®), Lormetazepam, Medazepam, Midazolam, Nitrazepam,Nordiazepam, Prazepam, Temazepam, Tetrazepam Cannabis Δ9-THC,11-Nor-Δ8-THC-9-COOH, 11-Nor-Δ9-THC-9-COOH, 11- (Marijuana, etc.)Hydroxy-Δ9-tetrahydrocannabinol, Δ8-Tetrahydrocannabinol, Δ9-Tetrahydrocannabinol, Cannabinol, Cannabidiol, pentanoic acid, butanoicacid, 4-hydroxybutyl, 4-hydroxypentyl Codeine Morphine, Codeine,Diacetyl morphine (heroine), Ethylmorphine, Hydromorphone, Meperidine,6-Monoacetylmorphine, Morphine-3- glucuronide, Oxycodone, Oxymorphone,Promethazine, Rifampicine, Thebaine, Trimipamine Nicotine/CotinineCotinine, Nicotine Morphine Morphine Tricyclic Nortriptyline,Amitriptyline, Chlorpromazine, Clomipramine, antidepressantsCyclobenzaprine, Desipramine, Diphenyldramine, Doxepine, Imipramine,(TCA's) Nordoxepine, Opipramol, Protriptyline, Perphenazine, Promazine,Promethazine, Trimipramine LSD LSD Methadone EDDP, Doxylamine,Methadone, Methadol Methaqualone Methaqualone, 3-hydroxy methaqualone,4-hydroxy methaqualone, 2- hydroxy methaqualone, Amitriptyline,Carbamazepine, Nortriptyline, Phenytoin, Primidone, Theophylinebuprenorphine Buprenorphine, Buprenorphine-3-B-d-gluconoride,Nor-Buprenorphine, Nor-Buprenorphine-3-B-d-gluconoride KetamineKetamine, Norketamine, Dextromethorphan, Dextrorphantartrate, EDDP,Phencyclidine, Promazine, Meperidine, D-Methamphetamine, Mephentermineh. s., MDEA, Nordoxepin hydrochloride, Promethazine, D- Norpropoxyphene,Methadone MethCathinone MethCathinone, 4-MMC (Mephedrone), 3-MMC(3-methylmethcathinone), 4-MEC (4-methylethcathinone), Methylone (MDMC,bk-MDMA), Cathinone, MDPV MDPV MDPV, Cathinone, MethCathinonemethylphenidate methylphenidate tramadol Tramadol, N-demethyl-tramadol,O-demethyl-tramadol oxycodone Oxycodone, Oxymorphone, Codeine, DiacetylMorphine (Heroine), Ethylmorphine, Hydrocodone, Hydromorphone,Merperidine, 6- Monoacetylmorphine, Morphine,Morphine-3-beta-D-glucuronide, Thebaine propoxyphene D-propoxyphene,D-norpropoxyphene Fentanyl Methaqualone, Mecloqualone, 3-hydroxymethaqualone, 4-hydroxy methaqualone, 2-hydroxy methaqualone,Amitriptyline, Carbamazepine, Nortriptyline, Phenytoin, Primidone,Theophyline 10. Coagulation Disorders Congenital Platelet, Fibronogen,Factor V, Anti-Xa, FactorXIII screen, D-dimer hemophilia; Von Willebranddisease; Acquired hemophilia 11. Fecal Occult Blood Test Colon Cancer;Blood cells, Hemoglobin, Fecal DNA colon polyps; crohn's disease;hemorrhoids; anal fissures; intestinal infections; Ulcers; Ulcerativecolitis 12. Blood Gas and Electrolytes pH, pCO₂, pO₂, Sodium (Na+),Potassium (K+), Calcium (Ca++), HCO3, TCO2, SBE

In some embodiments, the devices, systems and methods of the inventioncan be used to detect or diagnose a health condition. In someembodiments, the health condition includes, but is not limited to:chemical balance; nutritional health; exercise; fatigue; sleep; stress;prediabetes; allergies; aging; exposure to environmental toxins,pesticides, herbicides, synthetic hormone analogs; pregnancy; menopause;and andropause.

In some embodiments, relative levels of nucleic acids in two or moredifferent nucleic acid samples can be obtained using such methods, andcompared. In these embodiments, the results obtained from the methodsdescribed herein are usually normalized to the total amount of nucleicacids in the sample (e.g., constitutive RNAs), and compared. This can bedone by comparing ratios, or by any other means. In particularembodiments, the nucleic acid profiles of two or more different samplescan be compared to identify nucleic acids that are associated with aparticular disease or condition.

In some embodiments, the devices, systems and methods in the presentinvention can include a) obtaining a sample, b) applying the sample todevice containing a capture agent that binds to an analyte of interest,under conditions suitable for binding of the analyte in a sample to thecapture agent, c) washing the device, and d) reading the device, therebyobtaining a measurement of the amount of the analyte in the sample. Insome embodiments, the analyte can be a biomarker, an environmentalmarker, or a foodstuff marker. The sample in some instances is a liquidsample, and can be a diagnostic sample (such as saliva, serum, blood,sputum, urine, sweat, lacrima, semen, or mucus); an environmental sampleobtained from a river, ocean, lake, rain, snow, sewage, sewageprocessing runoff, agricultural runoff, industrial runoff, tap water ordrinking water; or a foodstuff sample obtained from tap water, drinkingwater, prepared food, processed food or raw food. In some embodiments,the device can be placed in a microfluidic device and the applying stepb) can include applying a sample to a microfluidic device comprising thedevice. In some embodiments, the reading step d) can include detecting afluorescence or luminescence signal from the device. In someembodiments, the reading step d) can include reading the device with ahandheld device configured to read the device. The handheld device canbe a mobile phone, e.g., a smart phone. In some embodiments, the devicecan include a labeling agent that can bind to an analyte-capture agentcomplex on the device. In some embodiments, the devices, systems andmethods in the present invention can further include, between steps c)and d), the steps of applying to the device a labeling agent that bindsto an analyte-capture agent complex on the device, and washing thedevice. In any embodiment, the reading step d) can include reading anidentifier for the device. The identifier can be an optical barcode, aradio frequency ID tag, or combinations thereof. In some embodiments,the devices, systems and methods in the present invention can furtherinclude applying a control sample to a control device containing acapture agent that binds to the analyte, wherein the control sampleincludes a known detectable amount of the analyte, and reading thecontrol device, thereby obtaining a control measurement for the knowndetectable amount of the analyte in a sample. In some embodiments, thesample can be a diagnostic sample obtained from a subject, the analytecan be a biomarker, and the measured amount of the analyte in the samplecan be diagnostic of a disease or a condition.

In some embodiments, the devices, systems and methods in the presentinvention can further include receiving or providing to the subject areport that indicates the measured amount of the biomarker and a rangeof measured values for the biomarker in an individual free of or at lowrisk of having the disease or condition, wherein the measured amount ofthe biomarker relative to the range of measured values is diagnostic ofa disease or condition. In some embodiments, the devices, systems andmethods in the present invention can further include diagnosing thesubject based on information including the measured amount of thebiomarker in the sample. In some embodiments, the diagnosing stepincludes sending data containing the measured amount of the biomarker toa remote location and receiving a diagnosis based on informationincluding the measurement from the remote location. In some embodiments,the biomarker can be selected from those listed in the Tables. In someembodiments, the device can contain a plurality of capture agents thateach binds to a biomarker described herein, wherein the reading step d)includes obtaining a measure of the amount of the plurality ofbiomarkers in the sample, and wherein the amount of the plurality ofbiomarkers in the sample is diagnostic of a disease or condition. Insome embodiments, the capture agent can be an antibody epitope and thebiomarker can be an antibody that binds to the antibody epitope. In someembodiments, the antibody epitope includes a biomolecule, or a fragmentthereof, selected from the Tables. In some embodiments, the antibodyepitope includes an allergen, or a fragment thereof, selected from theTables. In some embodiments, the antibody epitope includes an infectiousagent-derived biomolecule, or a fragment thereof, selected from Tables.In some embodiments, the device can contain a plurality of antibodyepitopes selected from the Tables, wherein the reading step d) includesobtaining a measure of the amount of a plurality of epitope-bindingantibodies in the sample, and wherein the amount of the plurality ofepitope-binding antibodies in the sample is diagnostic of a disease orcondition.

In some embodiments, the sample can be an environmental sample, andwherein the analyte can be an environmental marker. In some embodiments,the environmental marker described herein. In some embodiments, themethod can include receiving or providing a report that indicates thesafety or harmfulness for a subject to be exposed to the environmentfrom which the sample was obtained. In some embodiments, the method caninclude sending data containing the measured amount of the environmentalmarker to a remote location and receiving a report that indicates thesafety or harmfulness for a subject to be exposed to the environmentfrom which the sample was obtained. In any embodiment, the device caninclude a plurality of capture agents that each binds to anenvironmental marker described herein, and wherein the reading step d)can include obtaining a measure of the amount of the plurality ofenvironmental markers in the sample.

In some embodiments, the sample can be a foodstuff sample, wherein theanalyte can be a foodstuff marker, and wherein the amount of thefoodstuff marker in the sample can correlate with safety of thefoodstuff for consumption. In some embodiments, the foodstuff marker isan example described herein. In any embodiment, the method can includereceiving or providing a report that indicates the safety or harmfulnessfor a subject to consume the foodstuff from which the sample isobtained. In any embodiment, the method can include sending datacontaining the measured amount of the foodstuff marker to a remotelocation and receiving a report that indicates the safety or harmfulnessfor a subject to consume the foodstuff from which the sample isobtained. In any embodiment, the device array can include a plurality ofcapture agents that each binds to a foodstuff marker described herein,wherein the obtaining can include obtaining a measure of the amount ofthe plurality of foodstuff markers in the sample, and wherein the amountof the plurality of foodstuff marker in the sample can correlate withsafety of the foodstuff for consumption.

In some embodiments, the subject device is part of a microfluidicdevice. In some embodiments, the subject devices, systems, and methodsare used to detect a fluorescence or luminescence signal. In someembodiments, the subject devices, systems, and methods include, or areused together with, a communication device, such as but not limited to:mobile phones, tablet computers and laptop computers. In someembodiments, the subject devices, systems, and methods include, or areused together with, an identifier, such as but not limited to an opticalbarcode, a radio frequency ID tag, or combinations thereof.

In some embodiments, the sample is a diagnostic sample obtained from asubject, the analyte is a biomarker, and the measured amount of theanalyte in the sample is diagnostic of a disease or a condition. In someembodiments, the subject devices, systems and methods further includereceiving or providing to the subject a report that indicates themeasured amount of the biomarker and a range of measured values for thebiomarker in an individual free of or at low risk of having the diseaseor condition, wherein the measured amount of the biomarker relative tothe range of measured values is diagnostic of a disease or condition.

In some embodiments, the sample is an environmental sample, and whereinthe analyte is an environmental marker. In some embodiments, the subjectdevices, systems and methods includes receiving or providing a reportthat indicates the safety or harmfulness for a subject to be exposed tothe environment from which the sample was obtained. In some embodiments,the subject devices, systems and methods include sending data containingthe measured amount of the environmental marker to a remote location andreceiving a report that indicates the safety or harmfulness for asubject to be exposed to the environment from which the sample wasobtained.

In some embodiments, the sample is a foodstuff sample, wherein theanalyte is a foodstuff marker, and wherein the amount of the foodstuffmarker in the sample correlate with safety of the foodstuff forconsumption. In some embodiments, the subject devices, systems andmethods include receiving or providing a report that indicates thesafety or harmfulness for a subject to consume the foodstuff from whichthe sample is obtained. In some embodiments, the subject devices,systems and methods include sending data containing the measured amountof the foodstuff marker to a remote location and receiving a report thatindicates the safety or harmfulness for a subject to consume thefoodstuff from which the sample is obtained.

Various samples can be used in the assays conducted with the devices,apparatus, and systems herein described. In some embodiments, the samplecomprises nucleic acids. In some embodiments, the sample comprisesproteins. In some embodiments, the sample carbohydrates. The currentdevices, apparatus, and systems can be used to rapidly change thetemperature of the sample and steadily maintain the temperature of thesample, providing a fast and cost-effective approach to process samples.In addition, various applications (e.g. assays) can be conducted withthe devices, apparatus, and systems herein described. Such applicationsinclude but are not limited to diagnostic testing, health monitoring,environmental testing, and/or forensic testing. Such applications alsoinclude but are not limited to various biological, chemical, andbiochemical assays (e.g. DNA amplification, DNA quantification,selective DNA isolation, genetic analysis, tissue typing, oncogeneidentification, infectious disease testing, genetic fingerprinting,and/or paternity testing).

In some embodiments, the “sample” can be any nucleic acid containing ornot containing samples, including but not limited to human bodilyfluids, such as whole blood, plasma, serum, urine, saliva, and sweat,and cell cultures (mammalian, plant, bacteria, fungi). The sample can befreshly obtained, or stored or treated in any desired or convenient way,for example by dilution or adding buffers, or other solutions orsolvents. Cellular structures can exist in the sample, such as humancells, animal cells, plant cells, bacteria cells, fungus cells, andvirus particles.

The term “nucleic acid” as used herein refers to any DNA or RNAmolecule, or a DNA/RNA hybrid, or mixtures of DNA and/or RNA. The term“nucleic acid” therefore is intended to include but not limited togenomic or chromosomal DNA, plasmid DNA, amplified DNA, cDNA, total RNA,mRNA and small RNA. The term “nucleic acid” is also intended to includenatural DNA and/or RNA molecule, or synthetic DNA and/or RNA molecule.In some embodiments, cell-free nucleic acids are presence in the sample,as used herein “cell-free” indicates nucleic acids are not contained inany cellular structures. In some other embodiments, nucleic acids arecontained within cellular structures, which include but not limited tohuman cells, animal cells, plant cells, bacterial cells, fungi cells,and/or viral particles. Nucleic acids either in the form of cell-freenucleic acids or within cellular structures or a combination thereof,can be presence in the sample. In some further embodiments, nucleicacids are purified before introduced onto the inner surface of the firstplate. In yet further embodiments, nucleic acids can be within a complexassociated with other molecules, such as proteins and lipids.

The method of the invention is suitable for samples of a range ofvolumes. Sample having different volumes can be introduced onto theplates having different dimensions.

As used herein, “nucleic acid amplification” includes any techniquesused to detect nucleic acids by amplifying (generating numerous copiesof) the target molecules in samples, herein “target” refers to asequence, or partial sequence, of nucleic acid of interest.

Suitable nucleic acid amplification techniques include but not limitedto, different polymerase chain reaction (PCR) methods, such as hot-startPCR, nested PCR, touchdown PCR, reverse transcription PCR, RACE PCR,digital PCR, etc., and isothermal amplification methods, such asLoop-mediated isothermal amplification (LAMP), strand displacementamplification, helicase-dependent amplification, nicking enzymeamplification, rolling circle amplification, recombinase polymeraseamplification, etc.

As used herein, “necessary reagents” or “reagents” include but are notlimited to, primers, deoxynucleotides (dNTPs), bivalent cations (e.g.Mg2+), monovalent cation (e.g. K+), buffer solutions, enzymes,additives, and reporters. “Necessary reagents for nucleic acidamplification” or “reagents for nucleic acid amplification” can beeither in the dry form on the inner surface of the first or the secondplate or both, or in a liquid form encased in, embedded in, orsurrounded by, a material that melts with increasing temperatures, suchas, for example, paraffin.

As used herein, “primers”, in some embodiments, can refer to a pair offorward and reverse primers. In some embodiments, primers can refer to aplurality of primers or primer sets. As used herein, enzymes suitablefor nucleic acid amplification include, but not limited to,DNA-dependent polymerase, or RNA-dependent DNA polymerase, orDNA-dependent RNA polymerase. Examples of suitable DNA-dependentpolymerases include but not limited to AptaTaq polymerase, Kapa2G Fastpolymerase, Kapa2G Robust, Z-Taq polyermase, Terra PCR DirectPolymerase, SpeedStar HS DNA polymerase, Phusion DNA polymerase, andHigh-Fidelity DNA polymerase.

As used herein, “additives”, in some embodiments, include but notlimited to, 7-deaza-2′-deoxyguanosine 7-deaza dGTP, BSA, gelatin,betaine, DMSO, formamide, Tween 20, NP-40, Triton X-100,tetramethylammonium chloride.

As used herein, the term “reporter” refers to any tag, label, or dyethat can bind to, or intercalate within, the nucleic acid molecule or beactivated by byproducts of the amplification process to enablevisualization of the nucleic acid molecule or the amplification process.Suitable reporters include but are not limited to fluorescent labels ortags or dyes, intercalating agents, molecular beacon labels, orbioluminescent molecules, or a combination thereof.

In some other embodiments, as used herein, “necessary reagents” or“reagents” (e.g., for nucleic acid amplification reactions) can alsoinclude cell lysing reagent, which facilitates to break down cellularstructures. Cell lysing reagents include but not limited to salts,detergents, enzymes, and other additives. The term “salts” hereininclude but not limited to lithium salt (e.g. lithium chloride), sodiumsalt (e.g. sodium chloride), potassium (e.g. potassium chloride). Theterm “detergents” herein can be ionic, including anionic and cationic,non-ionic or zwitterionic. The term “ionic detergent” as used hereinincludes any detergent which is partly or wholly in ionic form whendissolved in water. Suitable anionic detergents include but not limitedto sodium dodecyl sulphate (SDS) or other alkali metal alkylsulphatesalts or similar detergents, sarkosyl, or combinations thereof. The term“enzymes” herein include but not limited to lysozyme, cellulase, andproteinase. In addition, chelating agents including but not limited toEDTA, EGTA and other polyamino carboxylic acids, and some reducingagents, such as dithiotreitol (dTT), can also be included in cell lysingreagents. The compositions of necessary reagents herein vary accordingto rational designs of different amplification reactions. In someembodiments, for example when conducting isothermal amplification viaLAMP, the sample is heated to 60-65° C. for about 1-70 min.

As used herein, “nucleic acid amplification product” refers to variousnucleic acids generated by nucleic acid amplification techniques. Typesof nucleic acid amplification products herein include but not limited tosingle strand DNA, single strand RNA, double strand DNA, linear DNA, orcircular DNA, etc. In some embodiments, nucleic acid amplificationproduct can be identical nucleic acids having the same length andconfiguration. In some other embodiments, nucleic acid amplificationproducts can be a plurality of nucleic acids having different lengthsand configurations.

In some embodiments, nucleic acids accumulated after nucleic acidamplification is quantified using reporters. As defined and used above,reporter having quantifiable features that is correlated with thepresence or the absence, or the amount of the nucleic acid ampliconsaccumulated in the closed chamber.

As used herein, “cell lysing reagents”, intend to include but notlimited to salts, detergents, enzymes, and other additives, whichfacilitates to disrupt cellular structures. The term “salts” hereininclude but not limited to lithium salt (e.g. lithium chloride), sodiumsalt (e.g. sodium chloride), potassium (e.g. potassium chloride). Theterm “detergents” herein can be ionic, including anionic and cationic,non-ionic or zwitterionic. The term “ionic detergent” as used hereinincludes any detergent which is partly or wholly in ionic form whendissolved in water. Suitable anionic detergents include but not limitedto sodium dodecyl sulphate (SDS) or other alkali metal alkylsulphatesalts or similar detergents, sarkosyl, or combinations thereof. The term“enzymes” herein include but not limited to lysozyme, cellulase, andproteinase. In addition, chelating agents including but not limited toEDTA, EGTA and other polyamino carboxylic acids, and some reducingagents, such as dithiotreitol (dTT), can also be included in cell lysingreagents. The compositions of necessary reagents herein vary accordingto rational designs of different amplification reactions.

As used herein, “necessary reagent 2” include but not limited to,primers, deoxynucleotides (dNTPs), bivalent cations (e.g. Mg2+),monovalent cation (e.g. K+), buffer solutions, enzymes, and reporters.Necessary reagent 2 for nucleic acid amplification can be either in thedry form on the inner surface of the first or the second plate or both,or in a liquid form encased in, embedded in, or surrounded by, amaterial that melts with increasing temperatures, such as, for example,paraffin.

A Rapid Heating and Cooling Apparatus where a Separate Heating ElementOutside QMAX-Card

In some embodiments, the apparatus further comprises a separate heatingelement that is outside of RHC card and is configured to heat the RHCcard when being placed near or in contact with the RHC card. Theseparate heating element is capable of attaching or detaching a RHCcard, and gain energy from a heating source, in a similar fashion as theheating/cooling layer. The separate heating element allow a RHC cardwithout a heating/cooling layer. For example, as shown in FIGS. 36A and36B, the heating element is separate from the sample card.

The terms “CROF Card (or card)”, “COF Card”, “QMAX-Card”, “Q-Card”,“CROF device”, “COF device”, “QMAX-device”, “CROF plates”, “COF plates”,and “QMAX-plates” are interchangeable and may be used to identifyembodiments of the devices described herein. The term “X-plate” refersto one of the two plates in a CROF card, wherein the spacers are fixedto this plate. More descriptions of the COF Card, CROF Card, and X-plateare described in the provisional application Ser. No. 62/456,065, filedon Feb. 7, 2017, which is incorporated herein in its entirety for allpurposes.

A RHC card is a QMAX-care with or without spacer plus a heating/coolinglayer on or inside of one of the plate.

FIG. 26 shows a device card 100, which comprises a first plate 10 and asecond plate 20. In some embodiments, the first plate 10 and the secondplate 20 are moveable against each other into different configurations,including an open configuration and a closed configuration. In certainembodiments, in the open configuration, the two plates are partially orcompletely separated apart and the average spacing between the plates isat least 300 um. In certain embodiments, the sample can be deposited onone or both the plates. In certain embodiments, in the closedconfiguration, at least part of the sample is compressed by the twoplates into a layer, wherein the average sample thickness is 200 um orless.

In some embodiments, the QMAX card 100 comprises a hinge 103 thatconnects the first plate 10 and the second plate 20 so that the twoplates can pivot against each other. In some embodiments, the QMAX cardcomprises a notch 105, which facilitates the switching of the cardbetween the open configuration and the closed configuration. In someembodiments, one or both of the plates are transparent. In someembodiments, one or both of the plates are flexible. In someembodiments, the QMAX card 100 comprises a heating/cooling layer 190. Incertain embodiments, the heating/cooling layer 190 is configured toabsorb electromagnetic waves and convert the energy to increase thetemperature of the sample.

FIGS. 25A and 25B show perspective and sectional views of an embodimentof the device of the present invention. FIG. 25A illustrates the device(also termed “sample holder” of the system) 100 in an openconfiguration. As shown in FIG. 25A, the sample holder 100 comprises afirst plate 10, a second plate 20, and a spacing mechanism (not shown).The first plate 10 and second plate 20 respectively comprise an innersurface (11 and 21, respectively) and an outer surface (12 and 22,respectively). Each inner surface has a sample contact area (notindicated) for contacting a fluidic sample to be processed and/oranalyzed by the device.

The first plate 10 and the second plate 20 are movable relative to eachother into different configurations. One of the configurations is theopen configuration, in which, as shown in FIG. 25A, the first plate 10and the second plate 20 are partially or entirely separated apart, andthe spacing between the first plate 10 and the second plate 20 (i.e. thedistance between the first plate inner surface 11 and the second plateinner surface 21) is not regulated by the spacing mechanism. The openconfiguration allows a sample to be deposited on the first plate, thesecond plate, or both, in the sample contact area.

As shown in FIG. 25A, the second plate 20 further comprises aheating/cooling layer 112 in the sample contact area. It is alsopossible that the first plate 10 alternatively or additionally comprisethe heating/cooling layer 112. In some embodiments, the heating/coolinglayer 112 is configured to efficiently absorb radiation (e.g.electromagnetic waves) shed on it. The absorption percentage is 50% ormore, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more,99% or more, 100% or less, 85% or less, 75% or less, 65% or less, or 55%or less, or in a range between any of the two values. Theheating/cooling layer 112 is further configured to convert at least asubstantial portion of the absorbed radiation energy into heat (thermalenergy). For example, the heating/cooling layer 112 is configured toemit radiation in the form of heat after absorbing the energy fromelectromagnetic waves. The term “substantial portion” or “substantially”as used herein refers to a percentage that is 50% or more, 60% or more,70% or more, 80% or more, 90% or more, 95% or more, 99% or more, 99% ormore, or 99.9% or more.

FIGS. 24A and 24B illustrate the sample card in a closed configuration,where the heating/cooling layer comprises a heating zone that isdirectly being/to be heated by a heating source; FIG. 24A shows aprospective view and FIG. 24B shows a sectional view. In someembodiments, the heating/cooling layer comprises a heating zone that isbeing/to be directly heated by a heating source. In some embodiments,the heating sources emits electromagnetic radiation (waves) that, withor without modulation by lenses or other modulators, reaches theheating/cooling layer. The area that directly receives such radiation(waves) is referred to as the heating zone.

In some embodiments, the heating zone is smaller than the entire area ofthe heating/cooling layer. In some embodiments, the heating zone isabout 1/1000, 1/500, 1/200, 1/100, 1/50, 1/20, 1/10, 1/5, 1/2, or 2/3 ofthe area of the heating/cooling layer, or in a range between any of thetwo values. In some embodiments, when the sample is loaded andcompressed, by the two plates, into a thin layer, the volume of thesample directly in the path of the electromagnetic waves, or directly incontact with the area of the heating zone, is referred to as the heatedvolume. In some embodiments, since the sample layer is thin and/or dueto the superior absorption properties of the heating/cooling layer, thesample in the heated volume can be rapidly heated to a desiredtemperature. In some embodiments, the sample in the heated volume canalso be rapidly cooled to a desired temperature.

Biochemistry and Assays

The thermal cycler system and associated methods of the presentinvention can be used to facilitate a chemical, biological or medicalassay or reaction. In some embodiments, the reaction requirestemperature changes. In some embodiments, the reaction requires orprefers rapid temperature change in order to avoid non-specific reactionand/or reduce wait time. In certain embodiments, the system and methodsof the present invention is used to facilitate a reaction that requirescyclical temperature changes for amplification of a nucleotide in afluidic sample; such reactions include but are not limited to polymerasechain reaction (PCR). The descriptions below use PCR as an example toillustrate the capability and utilization of the thermal cycler systemand method of the present invention. It is should be noted, however,some embodiments of the device, systems and method herein described alsoapply to other assays and/or reactions that require temperature controland change.

In some embodiments, the assays (e.g. PCR) can be conducted with anon-processed sample. For example, the template of a PCR reaction can beprovided by a sample directed obtained from a subject without additionalprocessing. In some embodiments, the sample can be whole blood from anindividual. In some embodiments, such a “one-step” approach would allowfor more convenient use of the devices herein described.

In some embodiments, the sample 90 is a pre-mixed reaction medium forpolymerase chain reaction (PCR). For example, in certain embodiments,the reaction medium includes components such as but not limited to: DNAtemplate, two primers, DNA polymerase (e.g. Taq polymerase),deoxynucleoside triphosphates (dNTPs), bivalent cations (e.g. Mg²⁺),monovalent cation (e.g. K⁺), and buffer solution. The specificcomponents, the concentrations of each component, and the overall volumevaries according to rational design of the reaction. In someembodiments, the PCR assay requires a number of changes/alterations insample temperature between the following steps: (i) the optionalinitialization step, which requires heating the sample to 92-98° C.; (2)the denaturation step, which requires heating the sample to 92-98° C.;(3) the annealing step, which requires lowering the sample temperatureto 50-65° C.; (4) extension (or elongation) step, which requires heatingthe sample to 75-80° C.; (5) repeating steps (2)-(4) for about 20-40times;

and (6) completion of the assay and lowering the temperature of thesample to ambient temperature (e.g. room temperature) or cooling toabout 4° C. The specific temperature and the specific time period foreach step varies and depends on a number of factors, including but notlimited to length of the target sequence, length of the primers, thecation concentrations, and/or the GC percentage.

The thermal cycler system of the present invention provides rapidtemperature change for the PCR assay. For example, referring to panels(A) and (B) of FIG. 24 and panel (B) of FIG. 25 , in some embodiments,the sample 90 (e.g. pre-mixed reaction medium) is added to one or bothof the plates 10 and 20 in the open configuration and the plates isswitched to the closed configuration to compress the sample 90 into athin layer which has a thickness 102 that is regulated by a spacingmechanism (not shown); the heating source 202 projects anelectromagnetic wave 210 to the first plate 10 (e.g. specifically to theheating/cooling layer 112); the heating/cooling layer 112 is configuredto absorb the electromagnetic wave 210 and convert at least asubstantial portion of said electromagnetic wave 210 into heat, whichincreases the temperature of the sample; the removal of theelectromagnetic wave 210 results in a temperature decrease in the sample90.

In some embodiments, by projecting an electromagnetic wave 210 to theheating/cooling layer 112 or increasing the intensity of theelectromagnetic wave, the thermal cycler systems provide rapid heating(increase temperature) for any or all of the initialization step, thedenaturation step and/or the extension/elongation step; in someembodiments, with the removal of the electromagnetic wave projected fromthe heating source 202 or the decrease of the intensity of theelectromagnetic wave, the cooling to the annealing step and/or the finalcooling step is achieved with rapid speed. In some embodiments, theelectromagnetic wave 210 or an increase of the intensity of theelectromagnetic wave 210 creates an ascending temperature ramp rate ofat least 80° C./s, 70° C./s, 60° C./s, 50° C./s, 45° C./s, 40° C./s, 35°C./s, 30° C./s, 25° C./s, 20° C./s, 18° C./s, 16° C./s, 14° C./s, 12°C./s, 10° C./s, 9° C./s, 8° C./s, 7° C./s, 6° C./s, 5° C./s, 4° C./s, 3°C./s, or 2° C./s, or in a range between any of the two values. Incertain embodiments, the average ascending temperature ramp rate in aPCR assay is 10° C./s or more. In some embodiments, the removal of theelectromagnetic wave 210 or a reduction of the intensity of theelectromagnetic wave 210 results in a descending temperature ramp rateof at least 80° C./s, 70° C./s, 60° C./s, 50° C./s, 45° C./s, 40° C./s,35° C./s, 30° C./s, 25° C./s, 20° C./s, 18° C./s, 16° C./s, 1° C./s, 12°C./s, 10° C./s, 9° C./s, 8° C./s, 7° C./s, 6° C./s, 5° C./s, 4° C./s, 3°C./s, or 2° C./s, or in a range between any of the two values. Incertain embodiments, the average descending temperature ramp rate in aPCR assay is 5° C./s or more. As used here, the term “ramp rate” refersto the speed of temperature change between two pre-set temperatures. Insome embodiments, the average ascending or descending temperature toeach step is different.

During a PCR, within any step after the target temperature has beenreached, the sample needs to be maintained at the target temperature fora certain period of time. The thermal cycler system of the presentinvention provides the temperature maintenance function by (1) adjustingthe intensity of the electromagnetic wave 210, lowering it if thetemperature has been raised to the target or increasing it if thetemperature has been decreased to the target, and/or (2) keep the targettemperature by balancing the heat provided to the sample and the heatremoved from the sample.

FIG. 30 illustrates a cross-sectional view of an exemplary procedure fornucleic acid amplification using a device, according to someembodiments. Examples of steps include (A) introducing sample containingnucleic acids onto the inner side of a first plate (substrate); (B)pressing a second plate onto the inner surface of the first plate toform a closed configuration of the device, where necessary reagents fornucleic acid amplification are dried on the inner surface of the secondplate; (C) accumulating nucleic acid amplification products in thechamber enclosed by the first and the second plates.

The sample can be introduced onto either the first plate or the secondplate, or even both when necessary. FIG. 30 herein provides an exampleof introducing sample onto the first plate inner surface.

More particularly, in step (B), a second plate is pressed onto the innersurface of the first plate, in contact with the sample, to form a closedconfiguration of the device. A “a second plate” may refer to a platewith periodic spacers on the inner surface contacting samples.

More particularly, in step (C), when the device is in the closedconfiguration, a heating source projects an electromagnetic wave to theheating/cooling layer on the inner or outer surface of the first plate,or the second plate or both. The heating/cooling layer is configured toabsorb the electromagnetic wave and convert at least a substantialportion of the energy from the said electromagnetic wave into the formof heat, which transmitted to the sample in the closed chamber. In someembodiments, the heating source is programmed to adjust the temperatureof the said sample in a range from ambient temperature to 98° C. In someembodiments, for example for conventional PCR, the sample is firstheated to 98° C., and then undergoes a repeated cycle of 94° C., 50-65°C., and 72° C. for 15-40 times. In some embodiments, for example forisothermal amplification, the temperature of the sample is maintained ata constant temperature. In some embodiments, for example when conductingisothermal amplification via LAMP, the sample is heated to 60-65° C. forabout 1-70 min.

FIG. 31 illustrates a cross-sectional view of an exemplary assayprocedure combining nucleic acid extraction and amplification using acard device, according to some embodiments. Examples of steps include(A) immobilizing capture probes on the inner surface of a first plate(substrate); (B) introducing samples onto the inner surface of the firstplate; (C) pressing a second plate onto the inner surface of the firstplate to form a closed configuration of the device, where necessaryreagents 1 to facilitate releasing and capturing nucleic acids are driedon the inner surface of the second plate; (D) capturing nucleic acidsfrom the above said sample onto the inner surface of the first plate;(E) detaching the second plate and cleaning the inner surface of thefirst plate using sponge; (F) pressing a third plate onto the innersurface of the first plate, where necessary reagents 2 for nucleic acidamplification are dried on the inner surface of the third plate; (G)accumulating nucleic acid amplification products in the chamber enclosedby the first and the third plate.

In some embodiments, in step (a), capture probes are immobilized on theinner surface of the first plate. As used herein, “capture probes” mayrefer to oligonucleotides having the length between 1-200 bp, preferablybetween 5-50 bp, more preferably between 10-20 bp. Capture probes mayhave complementary sequence to nucleic acid sequences of interest in thesample. In some embodiments, identical capture probes may be immobilizedon the surface of the first plate. In some other embodiments, differentcapture probes having different base pair compositions may beimmobilized on the surface of the first plate. Capture probes can beDNA, or RNA, or both, but preferably to be single strand DNA. As usedherein, “immobilize” refers to a process to anchor the capture probe onthe plate surface. In some embodiments, capture probes are anchoredthrough covalent bond, wherein, for example, either 5′ or 3′ end of thecapture probe is modified to facilitate coating on the plate surface.Commonly used 3′ end modifications include but not limited to thiol,dithiol, amine, biotin, etc. In some other embodiments, capture probescan be passively absorbed on the substrate surface.

After immobilized with capture probes, the plate surface may be blockedwith blocker solutions. Suitable blockers include but not limited to6-Mercapto-hexanol, bovine serum albumin, etc.

As shown in step (B) in FIG. 31 , the “sample” can be any nucleic acidcontaining or not containing samples, including but not limited to humanbodily fluids, such as whole blood, plasma, serum, urine, saliva, andsweat, and cell cultures (mammalian, plant, bacteria, fungi), accordingto some embodiments. The sample can be freshly obtained, or stored ortreated in any desired or convenient way, for example by dilution oradding buffers, or other solutions or solvents. Cellular structures canexist in the sample, such as human cells, animal cells, plant cells,bacteria cells, fungus cells, and virus particles.

The sample can be introduced onto either the first plate or the secondplate, or even both when necessary. FIG. 31 herein provides an exampleof introducing sample onto the first plate inner surface.

In some embodiments, in step (C), a second plate is pressed onto theinner surface of the first plate (substrate), in contact with thesample, to form a closed configuration of the device. Necessary reagents1 for nucleic acid amplification can be either in the dry form on theinner surface of the first or the second plate or both, or in a liquidform encased in, embedded in, or surrounded by, a material that meltswith increasing temperatures, such as, for example, paraffin.

In some embodiments, in step (D), after in contact with the above saidsample, dried necessary reagent 1 dissolves in the sample. Nucleic acidsof interest, either released from disrupted cellular structures orpresence as cell-free nucleic acids, or a combination thereof, hybridizeto the complementary capture probes on the plate surface. Time used forhybridization varies, largely depending on the specifications of thespacers on the inner surface of the plate. In some embodiments, forexample, when a plate having 30 um spacers in height is used,experimental data indicated after 2 min, hybridization between nucleicacids of interest and immobilized capture probes reached equilibrium. Asused herein, “unhybridized nucleic acids” refer to nucleic acids thatare not captured by the immobilized capture probes.

In some embodiments, in step (E) of FIG. 31 , the second plate isdetached from the first plate (substrate) and the surface of the firstplate (substrate) is cleaned using sponge. As used herein, “sponge”refers to a class of flexible porous materials that change pore sizesunder different pressures. Sponges containing washing buffer are incontact with the first plate surface to remove contaminates. In someembodiments, sponges are in contact with the first plate surface for onetime. In some other embodiments, sponges are in contact with the firstplate surface for twice, or more than twice. As used herein,“contaminates” refer to compounds including but not limited to celldebris, proteins, non-specific nucleic acid, etc. that are detrimentalto the nucleic acid amplification reaction.

In some embodiments, in step (F) of FIG. 31 , a third plate (QMAX card2) is pressed onto the inner surface of the first plate, in contact withthe sample, to form a closed configuration of the device. Necessaryreagent 2 for nucleic acid amplification can be either in the dry formon the inner surface of the first or the third plate or both, or in aliquid form encased in, embedded in, or surrounded by, a material thatmelts with increasing temperatures, such as, for example, paraffin.

In some embodiments, in step (G) of FIG. 31 , when the device is in theclosed configuration, a heating source projects an electromagnetic waveto the heating/cooling layer on the inner or outer surface of the firstplate, or the third plate or both. The heating/cooling layer isconfigured to absorb the electromagnetic wave and convert at least asubstantial portion of the energy from the said electromagnetic waveinto the form of heat, which transmitted to the sample in the closedchamber. In some embodiments, the heating source is programmed to adjustthe temperature of the said sample in a range from ambient temperatureto 98° C. In some embodiments, for example for conventional PCR, thesample is first heated to 98° C., and then undergoes a repeated cycle of94° C., 50-65° C., and 72° C. for 15-40 times. In some embodiments, forexample for isothermal amplification, the temperature of the sample ismaintained at a constant temperature. In some embodiments, for examplewhen conducting isothermal amplification via LAMP, the sample is heatedto 60-65° C. for about 1-70 min.

In some embodiments, the sample contact area of one or both of theplates comprises a compressed open flow monitoring surface structures(MSS) that are configured to monitoring how much flow has occurred afterCOF. For examples, the MSS comprises, in some embodiments, shallowsquare array, which will cause friction to the components (e.g. bloodcells in a blood) in a sample. By checking the distributions of somecomponents of a sample, one can obtain information related to a flow,under a COF, of the sample and its components.

The depth of the MSS can be 1/1000, 1/100, 1/100, 1/5, 1/2 of the spacerheight or in a range of any two values, and in either protrusion or wellform.

Multiplexing

FIGS. 29A and 29B show perspective views of a sample holder 100 in anopen configuration (FIG. 29A) and a closed configuration (FIG. 29B),where there are multiple sample contact areas on the plates, allowingthe processing and analysis of multiple samples. As shown in FIGS. 29Aand 29B, the thermal cycler system of the present invention comprises asample holder 100 and a thermal control unit 200; the sample holder 100comprises a plurality of first plates 10, a second plate 20, and aplurality of spacing mechanisms (not shown); the thermal control unit200 comprises a heating source 202 and a controller 204.

Referring to FIG. 29A, one or both of the plates (e.g. the second plate20) comprises a plurality of sample contact areas (not marked). In someembodiments, one or both of the plates (e.g. the second plate 20)comprises a plurality of heating/cooling layers 112. FIG. 29A shows thesample holder 100 in an open configuration, in which the first plates 10and the second plate 20 are partially or entirely separated apart,allowing the deposition of one or more samples on one or both of theplates. In the open configuration, the spacing between the first plates10 and the second plate 20 is not regulated by the spacing mechanisms.

FIG. 29B shows the sample holder 100 in a closed configuration, in whichthe inner surfaces of the two plates face each other and the spacing 102between the two plates are regulated by the spacing mechanism (notshown). If one or more samples have been deposited on the plates, theplates are configured to compress each sample into a layer, thethickness of the layer is regulated by the spacing mechanism.

As shown in FIG. 29B, a plurality of first plates 10 is used to coverpart of the second plate 20. For example, each first plate 10 covers asingle sample contact area, onto which a sample is deposited. A spacingmechanism is present for each sample contact area and the spacingmechanisms have different heights, resulting in different spacing 102for each sample contact area and for different thickness for each samplelayer. For example, the spacing mechanism is pillar shaped spacers; eachsample contact area has a set of spacers having a uniform height;different sets of spacers have the same or different heights, resultingin same or different sample layer thickness for the different samples.

Referring to FIGS. 29A and 29B, in some embodiments, the controller 204directs the heating source 202 to project an electromagnetic wave 210 tothe second plate 20 (and thus the heating/cooling layer 112), where theelectromagnetic wave 210 is absorbed by the heating/cooling layer 112and converted to heat, resulting in change of temperature in thesamples. In some embodiments, when there are multiple sample contactareas, multiple samples are processed and analyzed. For example, incertain embodiments each of the sample is a pre-mixed PCR reactionmedium having different components. One sample holder 100 is used totest different conditions for amplifying the same nucleotide and/oramplifying different nucleotides with the same or different conditions.

Additional Exemplary Embodiments

AAA-1.1 A device for rapidly changing the temperature of a fluidicsample, comprising:

a first plate (10), a second plate (20), a heating layer (112-1), and acooling layer (112-2), wherein:

each of the first plate and the second plate has, on its respectiveinner surface, a sample contact area for contacting a fluidic sample;wherein the sample contact areas face each other, are separated by anaverage separation distance of 200 um or less between them, and arecapable of contacting the sample and sandwiching the sample betweenthem;

the heating layer is:

positioned on the inner surface, the outer surface, or inside of one ofthe plates, and

configured to heat a relevant volume of the sample, wherein the relevantvolume of the sample is a portion or an entirety of the sample that isbeing heated to a desired temperature; and

the cooling layer is:

positioned on the inner surface, the outer surface, or inside of one ofthe plates; and

configured to cool the relevant sample volume; and

comprises a layer of material that that has a thermal conductivity tothermal capacity ratio of 0.6 cm²/sec or larger;

wherein the distance between the cooling layer and a surface of therelevant sample volume is zero or less than a distance that isconfigured to make the thermal conductance per unit area between thecooling layer and the surface of the relevant sample volume equal to 70W/(m²·K) or larger; and

wherein, in some embodiments, the heating layer and cooling layer arethe same material layer that has a heating zone and a cooling zone, andwherein the heating zone and cooling zone can have the same area ordifferent areas.

AAA-1.2 A device for rapidly changing the temperature of a fluidicsample, comprising:

A first plate (10), a second plate (20), a heating layer (112-1), and acooling layer (112-2), wherein:

each of the first plate and the second plate has, on its respectiveinner surface, a sample contact area for contacting a fluidic sample;wherein the sample contact areas face each other, are separated by anaverage separation distance of 200 um or less from each other, and arecapable of contacting the sample and sandwiching the sample betweenthem;

the heating layer is:

positioned on the inner surface, the outer surface, or inside of one ofthe plates, and

configured to heat a relevant volume of the sample, wherein the relevantvolume of the sample is a portion or an entirety of the sample that isbeing heated to a desired temperature; and

the cooling layer is:

positioned on the inner surface, the outer surface, or inside of one ofthe plates; and

configured to cool the relevant sample volume; and

comprises a layer of material that that has a thermal conductivity tothermal capacity ratio of 0.6 cm²/sec or larger, wherein the highthermal conductivity to thermal capacity ratio layer has an area largerthan the lateral area of the sample volume;

wherein the distance between the cooling layer and a surface of therelevant sample volume is zero or less than a distance that isconfigured to make the thermal conductance per unit area between thecooling layer and the surface of the relevant sample volume equal to 70W/(m²·K) or larger; and

wherein, in some embodiments, the heating layer and cooling layer arethe same material layer that has a heating zone and a cooling zone, andwherein the heating zone and cooling zone can have the same area ordifferent areas.

AAA-1.3 A device for rapidly changing the temperature of a fluidicsample, comprising:

a first plate (10), a second plate (20), a heating layer (112-1), and acooling layer (112-2), wherein:

the first and second plates are movable relative to each other intodifferent configurations;

each of the first plate and the second plate has, on its respectiveinner surface, a sample contact area for contacting a fluidic sample;wherein the sample contact areas face each other, are separated by anaverage separation distance of 200 um or less, and are capable ofsandwiching the sample between them;

the heating layer is:

positioned on the inner surface, the outer surface, or inside of one ofthe plates, and

configured to heat a relevant volume of the sample, wherein the relevantvolume of the sample is a portion or an entirety of the sample that isbeing heated to a desired temperature; and

the cooling layer is:

positioned on the inner surface, the outer surface, or inside of one ofthe plates; and

configured to cool the relevant sample volume; and

comprises a layer of material that that has a thermal conductivity tothermal capacity ratio of 0.6 cm²/sec or larger;

wherein the distance between the cooling layer and a surface of therelevant sample volume is zero or less than a distance that isconfigured to make the thermal conductance per unit area between thecooling layer and the surface of the relevant sample volume equal to 70W/(m²·K) or larger;

wherein one of the configurations is an open configuration, in which:the two plates are partially or completely separated apart and theaverage spacing between the plates is at least 300 um;

wherein another of the configurations is a closed configuration which isconfigured after the fluidic sample is deposited on one or both of thesample contact areas in the open configuration; and in the closedconfiguration: at least part of the sample is confined by the two platesinto a layer, wherein the average sample thickness is 200 um or less;and

wherein, in some embodiments, the heating layer and cooling layer arethe same material layer that has a heating zone and a cooling zone, andwherein the heating zone and cooling zone can have the same area ordifferent areas.

AAA-1.4 A device for rapidly changing the temperature of a fluidicsample, comprising:

a first plate (10), a second plate (20), spacers, a heating layer(112-1), and a cooling layer (112-2), wherein:

the first and second plates are movable relative to each other intodifferent configurations;

each of the first plate and the second plate has, on its respectiveinner surface, a sample contact area for contacting a fluidic sample;wherein the sample contact areas face each other, are separated by anaverage separation distance of 200 um or less between them, and arecapable of contacting the sample and sandwiching the sample betweenthem;

one or both of the plates comprise the spacers and the spacers are fixedon the inner surface of a respective plate;

the spacers have a predetermined substantially uniform height that isequal to or less than 200 microns, and the inter-spacer-distance ispredetermined;

the heating layer is:

positioned on the inner surface, the outer surface, or inside of one ofthe plates, and

configured to heat a relevant volume of the sample, wherein the relevantvolume of the sample is a portion or an entirety of the sample that isbeing heated to a desired temperature; and

the cooling layer is:

positioned on the inner surface, the outer surface, or inside of one ofthe plates; and

configured to cool the relevant sample volume; and

comprises a layer of material that that has a thermal conductivity tothermal capacity ratio of 0.6 cm²/sec or larger;

wherein the distance between the cooling layer and a surface of therelevant sample volume is zero or less than a distance that isconfigured to make the thermal conductance per unit area between thecooling layer and the surface of the relevant sample volume equal to 70W/(m²·K) or larger;

wherein one of the configurations is an open configuration, in which:the two plates are partially or completely separated apart, the spacingbetween the plates is not regulated by the spacers, and the sample isdeposited on one or both of the plates; and

wherein another of the configurations is a closed configuration which isconfigured after the sample is deposited in the open configuration; andin the closed configuration: at least part of the sample is compressedby the two plates into a layer of highly uniform thickness, wherein theuniform thickness of the layer is confined by the sample contactsurfaces of the plates and is regulated by the plates and the spacers;and

wherein, in some embodiments, the heating layer and cooling layer arethe same material layer that has a heating zone and cooling zone, andwherein the heating zone and cooling zone can have the same area ordifferent areas.

AAA-1.5 A device for rapidly changing the temperature of a fluidicsample, comprising:

a first plate (10), a second plate (20), and a heating/cooling layer(112), wherein:

the first plate (10) and the second plate (20) face each other, and areseparated by a distance from each other;

each of the plates has, on its respective inner surface (11, 21), asample contact area for contacting a fluidic sample; wherein the samplecontact areas are facing each other, are in contact with the sample,sandwich a sample between them, and have an average separation distance(102) from each other,

the heating/cooling layer (112) is on the outer surface (22) of thesecond plate (20); and

the heating/cooling layer is configured to comprise a heating zone and acooling zone; wherein the heat zone is configured to heat the fluidicsample, the cooling zone is configured to cool the sample significantlyby thermal radiative cooling;

wherein the heating zone is configured to receive a heating energy froma heating source and to have an area smaller than the total area of theheating/cooling layer; and

wherein at least a part of a heating zone of the heating layer overlapswith the sample area.

AAA-1.6 A device for rapidly changing the temperature of a fluidicsample, comprising:

a first plate (10), a second plate (20), and a heating/cooling layer(112), wherein:

each of the first plate (10) and the second plate (20) has, on itsrespective inner surface (11, 21), a sample contact area for contactinga fluidic sample; wherein the sample contact areas are facing eachother, are separated by an average separation distance (102) from eachother, and are capable of contacting the sample and sandwiching thesample between them;

the heating/cooling layer (112) has a thermal conductivity of 50 W/(m·K)or larger and is on the outer surface (22), on the inner surface, orinside of the second plate (20); and

the heating/cooling layer is configured to comprise a heating zone and acooling zone; wherein the heating zone is configured to heat a portionof the sample and have an area smaller than the total area of theheating/cooling layer, and wherein the cooling zone is configured tocool the sample;

wherein the heating zone, the second plate, and the portion of thesample are configured to have a scaled thermal conduction ratio (STCratio) of 2 or larger;

wherein the heating zone is configured to receive a heating energy froma heating source; and wherein at least a part of the heating zone of theheating layer overlaps with the sample area.

AAA-1.7 A device for rapidly changing the temperature of a fluidicsample, comprising:

a first plate (10), a second plate (20), and a heating/cooling layer(112), wherein:

each of the plates has, on its respective inner surface (11, 21), asample contact area for contacting a fluidic sample; wherein the samplecontact areas are facing each other, are in contact with the sample,sandwich the sample between them, and have an average separationdistance (102) from each other;

the heating/cooling layer (112) has a thermal conductivity of 50 W/(m·K)or larger and is on the outer surface (22), on the inner surface, orinside of the second plate (20); and

the heating/cooling layer is configured to comprise a heating zone and acooling zone; wherein the heating zone is configured to heat a portionof the sample and have an area smaller than the total area of theheating/cooling layer, and wherein the cooling zone is configured tocool the sample;

wherein the heating zone, the second plate, and the portion of thesample are configured to have a scaled thermal conduction ratio (STCratio) of 2 or larger;

wherein the heating/cooling layer has a thermal conductivity multiplyingits thickness in the range of 6×10⁻⁵ W/K to 3×10⁻⁴ W/K.

wherein the heating zone is configured to receive a heating energy froma heating source; and

wherein at least a part of the heating zone of the heating layeroverlaps with the sample area.

AAA-2.1. The device of any prior embodiments, wherein the heating layeris configured to be heated by a heating source.

AAA-2.2. The device of any prior embodiments, wherein the heating layeris the same layer as the cooling layer, and the same layer comprises aheating zone area and a cooling zone area.

AAA-2.3. The device of any prior embodiments, wherein the heating layer(i.e. the heating zone) has an area smaller than the cooling layer (i.e.cooling zone).

AAA-2.4. The device of any prior embodiments, wherein the heating layer(i.e., the heating zone) has an area that is about 1/100, 1/50, 1/20,1/10, 1/8, 1/6, 1/5, 1/4, 1/3, 1/2, 2/3, 3/4 or 5/6 of the cooling layer(i.e. cooling zone) area, or in a range between any of the two values.

AAA-2.5. The device of any prior embodiments, wherein the distancebetween the cooling layer and a surface of the relevant sample volume iszero or less than a distance that is configured to make the thermalconductance per unit area between the cooling layer and the surface ofthe relevant sample volume equal to 150 W/(m²·K) or larger.

AAA-2.6. The device of any prior embodiments, wherein the heating layercomprises metallic plasmonic materials, metamaterials, black silicon,graphite, carbon nanotube, silicon sandwich, graphene, or superlattice,or a combination thereof.

AAA-2.7. The device of any prior embodiments, wherein the heating layercomprises Al, Ag, or Au, with or without a paint layer.

AAA-2.8. The device of any prior embodiments, wherein the heating layerhas a thermal conductance per unit area that is equal to or larger than1000 W/(m²·K), 2000 W/(m²·K), 3000 W/(m²·K), 4000 W/(m²·K), 5000W/(m²·K), 7000 W/(m²·K), 10000 W/(m²·K), 20000 W/(m²·K), 50000 W/(m²·K),50000 W/(m²·K), 100000 W/(m²·K), or in range between any of the twovalues.

AAA-2.9. The device of any prior embodiments, wherein the heating layerhas a thermal conductance per unit area that is in a range of 1000W/(m²·K) to 2000 W/(m²·K), 2000 W/(m²·K) to 4000 W/(m²·K), 4000 W/(m²·K)to 10,000 W/(m²·K), or 10000 W/(m²·K) to 100000 W/(m²·K).

AAA3.1 The device of any prior embodiments, wherein the cooling layerhas a thermal conductance per unit area that is equal to or larger than1000 W/(m²·K), 2000 W/(m²·K), 3000 W/(m²·K), 4000 W/(m²·K), 5000W/(m²·K), 7000 W/(m²·K), 10000 W/(m²·K), 20000 W/(m²·K), 50000 W/(m²·K),50000 W/(m²·K), 100000 W/(m²·K), or in range between any of the twovalues.

AAA-3.2. The device of any prior embodiments, wherein the cooling layerhas a thermal conductance per unit area that is in a range of 1000W/(m²·K) to 2000 W/(m²·K), 2,000 W/(m²·K) to 4,000 W/(m²·K), 4,000W/(m²·K) to 10,000 W/(m²·K), or 10,000 W/(m²·K) to 100,000 W/(m²·K).

AAA-3.3 The device of any prior embodiments, wherein the cooling layercools the relevant sample primarily by thermal radiative cooling.

AAA-3.4 The device of any prior embodiments, wherein the cooling of therelevant sample through thermal radiative cooling is larger than thecooling through thermal conduction cooling in the direction lateral tothe plates.

AAA-3.5 The device of any prior embodiments, wherein the cooling of thesample through thermal radiative cooling is at least 1.2 times, 1.5times, 2 times, 5 times, 10 times, times, 50 times, 100 times, 200times, 500 times, or 1000 times larger than the cooling through thermalconduction cooling, or in a range between any of the two values.

AAA4.1 The device of any prior embodiments, wherein the heating layer orthe cooling layer has a thickness that is about 0.1 um, 0.2 um, 0.5 um,1 um, 2 um, 5 um, 10 um, um, 30 um, 40 um, 50 um, 100 um, 200 um, 500um, 1 mm, 2 mm, 5 mm, 10 mm, 20 mm, or 50 mm, or in a range between anyof the two values.

AAA4.2 The device of any prior embodiments, wherein the heating layer orthe cooling layer has an area that is less than 0.01 mm², 0.02 mm², 0.05mm², 0.1 mm², 0.2 mm², 0.5 mm², 1 mm², 2 mm², 5 mm², 10 mm², 20 mm², 50mm², 100 mm², 200 mm², 500 mm², or 1000 mm², or in a range between anyof the two values.

AAA4.3 The device of any prior embodiments, wherein the heating layer orthe cooling layer has an area dimension that is about 1 mm, 2 mm, 3 mm,5 mm, 6 mm, 7 mm, 8 mm, 9 mm 10 mm, 12 mm, or 15 mm, or in a rangebetween any two values.

AAA4.4 The device of any prior embodiments, wherein the heating layer orthe cooling layer comprises metallic plasmonic materials, metamaterials,black silicon, graphite, carbon nanotube, silicon sandwich, graphene, orsuperlattice, or a combination thereof.

AAA5.1 The device of any prior embodiments, wherein the heating layerand the cooling layer are structurally separate layers, the heatinglayer has a heating zone, and the cooling layer has a cooling zone.

AAA6.1 The device of any prior embodiments, wherein the ratio of thecooling zone area to the heating zone area is larger than 1, 1.5, 2,2.5, 3, 5, 10, 15, 20, 25, 50, 75, 100, 200, 500, or 1000, or in a rangebetween any of the two values.

AAA6.2 The device of any prior embodiments, wherein the cooling zonearea is larger than the lateral area of the relevant sample volume by afactor that is equal to or large than 1.2 times, 1.5 times, 2 times, 5times, 10 times, 20 times, 50 times, 100 times, 200 times, 500 times, or1000 times larger than the cooling through thermal conduction cooling,or in a range between any of the two values.

AAA6.3 The device of any prior embodiments, wherein the cooling of thedevice has a thermal radiation cooling that, during a thermal cycling,is equal to or larger than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, or 99% of the total cooling or in a range between any of the twovalues, wherein the total cooling is the sum of radiative cooling andconductive cooling.

AAA6.4 The device of any prior embodiments, wherein the cooling of thedevice by thermal radiation through a high K cooling layer, during athermal cycling, is equal to or larger than 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 99% of the total cooling or in a range between any ofthe two values, wherein the total cooling is the sum of radiativecooling and conductive cooling.

AAA7.1 The device of any prior embodiments, wherein at least one of theplates is flexible.

AAA8.1 The device of any prior embodiments, wherein the device comprisesspacers that regulate the thickness of the sample when the sample isconfined by the two plates into a thin layer.

AAA8.2 The device of any prior embodiments, wherein the spacers has aninter-spacer-distance (ISD), and wherein the fourth power of theinter-spacer-distance (ISD) divided by the thickness (h) and the Young'smodulus (E) of the flexible plate (ISD4/(hE)) is 5×10⁶ um³/GPa or less.

AAA8.3. The device of any prior embodiments, wherein the spacers has acontact filling factor, wherein the product of the contact fillingfactor and the Young's modulus of the spacers is 2 MPa or larger, andwherein the contact filling factor is, in the sample contact area, theratio of the contact area between spacer and the plate to the totalplate area.

AAA8.4. The device of any prior embodiments, wherein the spacers are inthe sample contact area.

AAA8.5. The device of any prior embodiments, wherein the spacers have ashape of the pillars with substantially flat top.

AAA8.6. The device of any prior embodiments, wherein the spacers arefixed on either one or both of the plates.

AAA8.7. The device of any prior embodiments, wherein the spacers have auniform height.

AAA8.8. The device of any prior embodiments, wherein the thickness ofthe sample is the same as the height of the spacers.

AAA9.1 The device of any prior embodiments, wherein the heating layerand/or the cooling layer is on the inner surface of one of the plates.

AAA9.2 The device of any prior embodiments, wherein the heating layerand/or the cooling layer is on the outer surface of one of the plates.

AAA9.3 The device of any prior embodiments, wherein the heating layerand the cooling layer are separate, the heating layer is on the outersurface of one of the plates, and the cooling layer is on the outersurface of the other plate.

AAA9.4 The device of any prior embodiments, wherein the heating layerand the cooling layer are separate, the heating layer is on the innersurface of one of the plates, and the cooling layer is on the innersurface of the other plate.

AAA9.5 The device of any prior embodiments, wherein the heating layerand the cooling layer are separate, the heating layer is on the inner orouter surface of one of the plates, and the cooling layer is on theinner or outer surface of the other plate.

AAA9.6 The device of any prior embodiments, wherein the heating layerand the cooling layer are inside one or both of the plates.

AAA9.7 The device of any prior embodiments, wherein the heating zone andthe cooling zone are partly overlapping on the heating and/or coolinglayer.

AAA10.1 The device of any prior embodiments, wherein the first plate orthe second plate has a thickness that is less than 10 nm, 100 nm, 200nm, 500 nm, 1000 nm, 2 μm (micron), 5 μm, 10 μm, 20 μm, 50 μm, 100 μm,150 μm, 200 μm, 300 μm, 500 μm, 800 μm, 1 mm (millimeter), 2 mm, 3 mm, 5mm, 10 mm, 20 mm, 50 mm, 100 mm, 500 mm, or in a range between any twoof these values.

AAA10.2 The device of any prior embodiments, wherein the first plate orthe second plate has an lateral area that is less than 1 mm² (squaremillimeter), 10 mm², 25 mm², 50 mm², 75 mm², 1 cm² (square centimeter),2 cm², 3 cm², 4 cm², 5 cm², 10 cm², 100 cm², 500 cm², 1000 cm², 5000cm², 10,000 cm², 10,000 cm², or in a range between any two of thesevalues.

AAA10.3 The device of any prior embodiments, wherein the first plate orthe second plate comprises acrylate polymers, vinyl polymers, olefinpolymers, cellulosic polymers, noncellulosic polymers, polyesterpolymers, Nylon, cyclic olefin copolymer (COC), poly(methylmethacrylate) (PMMA), polycarbonate (PC), cyclic olefin polymer (COP),liquid crystalline polymer (LCP), polyimide (PA), polyethylene (PE),polyimide (PI), polypropylene (PP), poly(phenylene ether) (PPE),polystyrene (PS), polyoxymethylene (POM), polyether ether ketone (PEEK),polyether sulfone (PES), poly(ethylene phthalate) (PET),polytetrafluoroethylene (PTFE), polyvinyl chloride (PVC), polyvinylidenefluoride (PVDF), polybutylene terephthalate (PBT), fluorinated ethylenepropylene (FEP), perfluoroalkoxyalkane (PFA), polydimethylsiloxane(PDMS), rubbers, or any combinations of thereof.

AAA10.3.1 The device of any prior embodiments, wherein the first plateor the second plate comprises PMMA.

AAA10.4 The device of any prior embodiments, wherein the plates arethermal-isolated from a structure that accommodate the plates.

AAA11.1 The device of any prior embodiments, wherein the relevant samplehas a volume that is about 0.01 ul, 0.02 ul, 0.05 ul, 0.1 ul, 0.2 ul,0.5 ul, 1 ul, 2 ul, 5 ul, 10 ul, 20 ul, 50 ul, 100 ul, 200 ul, 500 ul, 1ml, 2 ml, 5 ml, 10 ml, 20 ml, or 50 ml, or in a range between any of thetwo values.

AAA11.2 The device of any prior embodiments, wherein ratio of thelateral average dimension of the relevant sample area to the samplethickness is greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50,60, 70, 80, 90, 100, 200, 500, 1000, 2000, 5000, 100,000, or in a rangebetween any of the two values.

AAA12.1 The device of any prior embodiments, wherein the plates areconfigured to be operable directly by human hands.

AAA12.2 The device of any prior embodiments, wherein the plates areconfigured to be compressed directly by human hands with impreciseforce, which is neither set to a precise level nor substantiallyuniform.

AAA12.3 The device of any prior embodiments, further comprising a hinge,which connects the first plate and the second plate and allows the twoplates to pivot against each other into different configurations.

AAA12.4 The device of any prior embodiments, wherein at least one of theplates comprises one or more open notches on an edge or corners of theplate, and the notch(es) facilitates changing the plates betweendifferent configurations.

AAA12.5 The device of any prior embodiments, wherein at least one of theplates comprises one or more open notches on an edge or corners of theplate, and the notch(es) facilitates changing the plates from aconfiguration that is near or at closed configuration to an openconfiguration.

AAA13.1. A sample cartridge, comprising the device of any priorembodiments, and a sample support that is configured to support thedevice.

AAA13.2. The sample cartridge of any prior embodiments, wherein thesample support comprises one or more apertures that allow energy toreach the heating layer.

AAA14.1 An apparatus for rapidly changing temperature of a fluidicsample, comprising:

the device of any prior embodiments;

a heating source that is configured to supply energy to the device.

AAA14.2 The apparatus of any prior embodiments, wherein the heatingsource is configured to radiate electromagnetic waves in a range ofwavelength that the heating/cooling layer has an absorption coefficientof 50% or higher.

AAA14.3. The apparatus of any prior embodiments, wherein the heatingsource comprises one or an array of light-emitting diodes (LEDs), one oran array of lasers, one or an array of lamps, or a combination ofthereof.

AAA14.4. The apparatus of any prior embodiments, wherein the heatingsource comprises halogen lamp, halogen lamp with reflector, LED withfocusing lens, laser with focusing lens, halogen lamp with couplingoptical fiber, LED with coupling optical fiber, laser with couplingoptical fiber.

AAA14.5 The apparatus of any prior embodiments, further comprising anoptical pipe between the heating source and the device, wherein theoptical pipe is configured to guide the energy from the heating sourceto the heating layer.

AAA15.1 An apparatus for rapidly changing temperature of a fluidicsample, comprising:

the device of any prior embodiments; and

an adaptor that is configured to accommodate the device.

AAA15.2 The apparatus of any prior embodiments, wherein the adaptorcomprises a sample slot that is configured to accommodate the device andposition the device to receive the electromagnetic waves from theheating source.

AAA15.3 The apparatus of any prior embodiments, wherein adaptorcomprises a slider that is configured to allow the device to slide intothe sample slot.

AAA16.1 An apparatus for rapidly changing temperature of a fluidicsample, comprising:

the device of any prior embodiments;

a heating source that is configured to supply energy the device; and

a control unit that is configured to control the heating unit.

AAA16.2 The apparatus of any prior embodiments, wherein the control unitis configured to control electromagnetic waves from the heating source.

AAA16.3 The apparatus of any prior embodiments, wherein the control unitis configured to control the presence, intensity, wavelength, frequency,and/or angle of the electromagnetic waves.

AAA16.2 The apparatus of any prior embodiments, wherein the control unitcomprises a temperature sensor that is configured to detect thetemperature of the sample.

A16.2.1 The apparatus of any prior embodiments, wherein the control unitcontrols the energy supplied by the heating source based on thetemperature detected by the temperature sensor.

AAA17.1. A system for rapidly changing temperature of a fluidic sample,comprising:

a device of any prior embodiments;

a heating source that is configured to emit electromagnetic waves thatcan be received by the device; and

a control unit, which is configured to control heating and cooling ofthe sample, at least in part by changing the electromagnetic waves fromthe heating source.

AAA17.2 The system of any prior embodiments, further comprising anadaptor that is configured to accommodate the device.

AAA17.3 The system of any prior embodiments, further comprising anoptical pipe that is configured to guide the electromagnetic waves fromthe heating source to the device.

AAA17.4 The system of any prior embodiments, further comprising a signalsensor that is configured to detect a signal from the sample.

AAA17.4.1. The system of any prior embodiments, wherein the signalsensor is an optical sensor that is configured to image the fluidicsample.

AAA18.1 A kit for rapidly changing temperature of a fluidic sample,comprising:

a device of any prior embodiments; and

reagents that configured to facilitate a chemical/biological reaction.

AAA18.2 The kit of any prior embodiments, wherein the reagents areconfigured for nucleic acid amplification:

AAA18.3 The kit of any prior embodiments, wherein the reagents comprisesa pre-mixed polymerase chain reaction (PCR) medium:

AAA18.4 The kit of any prior embodiments, wherein the reagents areconfigured to detect nucleic acids by amplifying (generating numerouscopies of) the target molecules in samples, wherein target moleculerefers to a sequence, or partial sequence, of nucleic acid of interest.

AAA18.5 The kit of any prior embodiments, wherein the reagents comprise:primers, deoxynucleotides (dNTPs), bivalent cations (e.g. Mg²⁺),monovalent cation (e.g. K⁺), buffer solutions, enzymes, or reporters, orany combination or mixture thereof.

AAA18.6 The kit of any prior embodiments, wherein the reagents areeither in the dry form on the inner surface of the first or the secondplate or both, or in a liquid form encased in, embedded in, orsurrounded by, a material that melts with increasing temperatures, suchas, for example, paraffin.

AAA18.7 The kit of any prior embodiments, wherein the reagents compriseDNA-dependent polymerase, or RNA-dependent DNA polymerase, orDNA-dependent RNA polymerase.

AAA18.8 The kit of any prior embodiments, wherein the reagents comprise“reporters” that refer to any tag, label, or dye that can bind to, orintercalate within, the nucleic acid molecule or be activated bybyproducts of the amplification process to enable visualization of thenucleic acid molecule or the amplification process.

AAA18.9 The kit of any prior embodiments, wherein the reagents comprisecell lysing reagent, which is configured to facilitate breaking downcellular structures.

AAA19.1 The device, apparatus, system, and/or kit of any priorembodiments, wherein the heating layer and/or the cooling layer areattached to the first plate and/or the second plate by e-beamevaporation.

AAA19.2. The device, apparatus, system, and/or kit of any priorembodiments, wherein the heating layer and/or the cooling layer comprisegold and the gold is attached to the first plate and/or the second plateby e-beam evaporation.

AA1. A device for rapidly changing a fluidic sample temperature,comprising:

a first plate, a second plate, and a heating/cooling layer, wherein:

the plates are movable relative to each other into differentconfigurations;

each of the plates has, on its respective inner surface, a samplecontact area for contacting a fluidic sample, and

the heating/cooling layer is configured to heat the fluidic sample;

wherein the heating/cooling layer is (a) on (either the inner or outersurface) or inside one of the plates, and (b) capable of being heated bya heating source, wherein the heating source delivers heat energy to theheating/cooling layer optically, electrically, by radio frequency (RF)radiation, or a combination thereof;

wherein at least a part of a heating area of the heating/cooling layeroverlaps with the sample area,

wherein one of the configurations is an open configuration, in which:the two plates are partially or completely separated apart and theaverage spacing between the plates is at least 300 um; and

wherein another of the configurations is a closed configuration which isconfigured after the fluidic sample is deposited on one or both of thesample contact areas in the open configuration; and in the closedconfiguration: at least part of the sample is compressed by the twoplates into a layer, wherein the average sample thickness is 200 um orless.

AA2.1 A device for rapidly changing temperature of a fluidic sample,comprising:

a sample holder and a heating/cooling layer, wherein:

the sample holder comprises a first plate and a second plate, whereineach of the plates comprises, on its respective surface, a samplecontact area for contacting the fluidic sample;

the first plate and the second plate are configured to confine thefluidic sample into a layer of highly uniform thickness of 0.1-200 umand substantially stagnant relative to the plates; and

the heating/cooling layer: (1) has a thickness of less than 1 mm, (2)has an area that is substantially less than the area of either the firstor the second plate, and (3) is configured to convert energy fromelectromagnetic waves into heat to raise the temperature of at leastpart of the fluidic sample in the layer of uniform thickness.

AA2.2 A device for rapidly changing temperature of a fluidic sample,comprising:

a sample holder and a heating/cooling layer, wherein:

the sample holder comprises a first plate and a second plate, whereineach of the plates comprises, on its respective surface, a samplecontact area for contacting the fluidic sample;

the first plate and the second plate are configured to confine at leastpart of the sample into a layer of highly uniform thickness of 0.1-200um and substantially stagnant relative to the plates,

the first plate has a thickness of 500 um or less, and the second platehas a thickness of 5 mm or less; and

the heating/cooling layer has a thickness of less than 1 mm and an areaof less than 100 mm² and is configured to convert energy fromelectromagnetic waves into heat to raise the temperature of the at leastpart of the fluidic sample in the layer of uniform thickness.

AA2.3 A device for rapidly changing temperature of a fluidic sample,comprising:

a sample holder and a heating/cooling layer, wherein:

the sample holder comprises a first plate and a second plate, whereineach of the plates comprises, on its respective surface, a samplecontact area for contacting the fluidic sample;

the first plate and the second plate are configured to confine at leastpart of the sample into a layer of highly uniform thickness of 0.1-200um and substantially stagnant relative to the plates,

the first plate has a thickness of 500 um or less, and the second platehas a thickness of 5 mm or less; and

the heating/cooling layer: (1) has a thickness of less than 1 mm, (2)has an area of less than 100 mm² that is substantially less than thearea of either the first or the second plate, and (3) is configured toconvert energy from electromagnetic waves into heat to raise thetemperature of at least part of the fluidic sample in the layer ofuniform thickness.

AA3 A device for rapidly changing temperature of a fluidic sample,comprising:

a sample holder and a heating/cooling layer, wherein:

the sample holder comprises a first plate and a second plate, whereineach of the plates comprises, on its respective surface, a samplecontact area for contacting the fluidic sample;

the first plate and the second plate are configured to confine at leastpart of the sample into a layer of highly uniform thickness of 500 um orless and substantially stagnant relative to the plates,

the first plate is in contact with the heating/cooling layer and has athickness of 1 um or less, and the second plate is not in contact withthe heating/cooling layer and has a thickness of 5 mm or less; and

the heating/cooling layer is configured to convert energy fromelectromagnetic waves into heat to raise the temperature of the at leastpart of the fluidic sample in the layer of uniform thickness, has anabsorption coefficient of 50% or higher, and has a thickness of lessthan 3 mm.

AA4 A device for rapidly changing temperature of a fluidic sample,comprising:

a sample holder and a heating/cooling layer, wherein:

the sample holder comprises a first plate and a second plate, whereineach of the plates comprises, on its respective surface, a samplecontact area for contacting the fluidic sample;

the first plate and the second plate are configured to confine at leastpart of the sample into a layer of highly uniform thickness of 500 um orless and substantially stagnant relative to the plates,

the first plate is in contact with the heating/cooling layer and has athickness of 1 um or less, and the second plate is not in contact withthe heating/cooling layer and has a thickness of 0.1-2 mm; and

the heating/cooling layer is configured to convert energy fromelectromagnetic waves into heat to raise the temperature of the at leastpart of the fluidic sample in the layer of uniform thickness, has anabsorption coefficient of 60% or higher, and has a thickness of lessthan 2 mm.

AA6.1 The device of any prior embodiments, wherein the heating/coolinglayer is on the inner surface of one of the plates.

AA6.2 The device of any prior embodiments, wherein the heating/coolinglayer is on the outer surface of one of the plates.

AA6.3 The device of any prior embodiments, wherein the heating/coolinglayer inside one of plates.

AA6.4 The device of any prior embodiments, wherein the heating/coolinglayer is in contact with at least one of the plates.

AA6.5 The device of any prior embodiments, wherein the heating/coolinglayer is not in contact with any of the plates.

AA6.6 The device of any prior embodiments, wherein the heating/coolinglayer is in contact with the sample when the plates are in the closedconfiguration.

AA7. The device of any prior embodiments, wherein the heating/coolinglayer is made from a single material or compound materials.

AA7.1 The device of any prior embodiments, wherein the heating/coolinglayer comprises semiconductors or metallic materials with high absorbingsurfaces.

AA7.2 The device of any prior embodiments, wherein the heating/coolinglayer comprises Silicon, Ge, InP, GaAs, CdTe, CdS, aSi, metal includingAu, Al, Ag, Ti, carbon coated Al, black painted Al, carbon (graphene,nanotube, nanowire) or a combination thereof.

AA7.3 The device of any prior embodiments, wherein the heating/coolinglayer is acting as the fast heating conductive layer comprises Silicon,Ge, InP, GaAs, CdTe, CdS, aSi, metal including Au, Al, Ag, Ti, carboncoated Al, black painted Al, carbon (graphene, nanotube, nanowire) or acombination thereof.

AA8 The device of any prior embodiments, wherein the part of the heatingarea that overlaps the sample area is less than 1%, 5%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the sample area, or in arange between any of the two values.

AA8.1 The device of any prior embodiments, wherein the part of theheating area that overlaps the sample area is less than 0.1 mm², 0.5mm², 1 mm², 5 mm², 10 mm², 25 mm², 50 mm², 75 mm², 1 cm² (squarecentimeter), 2 cm², 3 cm², 4 cm², 5 cm², 10 cm², or in a range betweenany of the two values.

AA9. The device of any prior embodiments, wherein the absorptioncoefficient of the heating/cooling layer is more than 30%, 40%, 50%,60%, 70%, 80%, 90%, or in a range between any of the two values.

AA9.1. The device of any prior embodiments, wherein the absorptioncoefficient of the heating/cooling layer is more than 60%, 70%, 80%,90%, or in a range between any of the two values.

AA9.2. The device of any prior embodiments, wherein the absorptioncoefficient of the heating/cooling layer is more than 60%.

AA10. The device of any prior embodiments, wherein the heating/coolinglayer has an absorption wavelength range that is 100 nm to 300 nm, 400nm to 700 nm (visible range), 700 nm to 1000 nm (IR range), 1 um to 10um, 10 um to 100 um, or in a range between any of the two values.

AA11. The device of any prior embodiments, wherein the heating/coolinglayer has a thickness equal to or less than 3 mm, 2 mm, 1 mm, 750 um,500 um, 250 um, 100 um, 50 um, 25 um, 10 um, 500 nm, 200 nm, 100 nm, or50 nm, or in a range between any of the two values.

AA12. The device of any prior embodiments, wherein the heating/coolinglayer has an area of 0.1 mm² or less, 1 mm² or less, 10 mm² or less, 25mm² or less, 50 mm² or less, 75 mm² or less, 1 cm² (square centimeter)or less, 2 cm² or less, 3 cm² or less, 4 cm² or less, 5 cm² or less, 10cm² or less, or in a range between any of the two values.

AA13. The device of any prior embodiments, wherein the first plate has athickness equal to or less than 500 um, 200 um, 100 um, 50 um, 25 um, 10um, 5 um, 2.5 um, 1 um, 500 nm, 400 nm, 300 nm, 200 nm, or 100 nm, or ina range between any of the two values.

AA13.1. The device of any prior embodiments, wherein the first plate hasa thickness equal of 10-200 um.

AA14. The device of any prior embodiments, wherein the second plate hasa thickness equal to or less than 5 mm, 4 mm, 3 mm, 2 mm, 1 mm, 750 um,500 um, 250 um, 100 um, 75 um, 50 um, or 25 um, or in a range betweenany of the two values.

AA14.1. The device of any prior embodiments, wherein the second platehas a thickness equal of 10-1000 um.

AA15. The device of any prior embodiments, wherein the sample layer hasa highly uniform thickness.

AA15.1 The device of any prior embodiments, wherein the sample layer hasa thickness of equal to or less than 100 um, 50 um, 20 um, 10 um, 5 um,1 um, 500 nm, 400 nm, 300 nm, 200 nm, or 100 nm, or in a range betweenany of the two values.

AA15.2. The device of any prior embodiments, wherein the sample layerhas a thickness of 1-100 um.

AA16. The device of any prior embodiments, wherein the area of at leastone of the plate is 1 mm² or less, 10 mm² or less, 25 mm² or less, 50mm² or less, 75 mm² or less, 1 cm² (square centimeter) or less, 2 cm² orless, 3 cm² or less, 4 cm² or less, 5 cm² or less, 10 cm² or less, 100cm² or less, 500 cm² or less, 1000 cm² or less, 5000 cm² or less, 10,000cm² or less, 10,000 cm² or less, or in a range between any two of thesevalues.

AA17.1 The device of any prior embodiments, wherein the area of at leastone of the plates is in the range of 500 to 1,000 mm²; or around 750mm².

AA18. The device of any prior embodiments, further comprising spacersthat are configured to regulate the thickness of the sample layer.

AA18.1 The device of any prior embodiments, wherein the spacers arefixed on either one or both of the plates.

AA18.2 The device of any prior embodiments, wherein the spacers arefixed on the inner surface of either one or both of the plates.

AA18.3 The device of any prior embodiments, wherein the spacers have auniform height.

AA18.4 The device of any prior embodiments, wherein at least one of thespacers is inside the sample contact area.

AA18.5 The device of any prior embodiments, wherein the thickness of thesample layer is the same as the height of the spacers.

AA19 The device any prior embodiments, wherein one or both plates areflexible.

AA20. The device of any prior embodiments, further comprising sealingstructures that are attached to either one or both of the contact andsecond plates, wherein the sealing structures are configured to limitthe evaporation of liquid inside the device.

AA21. The device of any prior embodiments, further comprising a clampingstructure that is attached to either one or both of the first and secondplates, wherein the clamp structure is configured to hold the device andregulate the thickness of the sample layer during the heating of thedevice.

AA22. The device of any prior embodiments, wherein the second plate istransparent for an electromagnetic wave from the sample.

AA23. The device of any prior embodiments, wherein the sample holder andthe heating/cooling layer are connected by a thermal coupler.

AA24. The device of any prior embodiments, wherein the areas of the atleast part of the sample and the heating/cooling layer are substantiallylarger than the uniform thickness.

AA25. The device of any prior embodiments, wherein the heating/coolinglayer is configured to absorb electromagnetic waves selected from thegroup consisting of: radio waves, microwaves, infrared waves, visiblelight, ultraviolet waves, X-rays, gamma rays, and thermal radiation.

AA26. The device of any prior embodiments, wherein the sample is apre-mixed polymerase chain reaction (PCR) medium.

AA27. The device of any prior embodiments, wherein the sample layer islaterally sealed to reduce sample evaporation.

AA28. The device of any prior embodiments, wherein the area of theradiation is smaller than the area of radiation absorption pad; The areaof the radiation absorption pad is less than the area of sample liquidarea; The area of sample liquid area is less than the first and secondplate size.

AA29. The device of any prior embodiments, wherein the fluidic samplecomprises a processed or unprocessed bodily fluid.

AA30. The device of any prior embodiments, wherein the fluidic samplecomprises amniotic fluid, aqueous humour, vitreous humour, blood (e.g.,whole blood, fractionated blood, plasma, serum, etc.), breast milk,cerebrospinal fluid (CSF), cerumen (earwax), chyle, chime, endolymph,perilymph, feces, gastric acid, gastric juice, lymph, mucus (includingnasal drainage and phlegm), pericardial fluid, peritoneal fluid, pleuralfluid, pus, rheum, saliva, sebum (skin oil), semen, sputum, sweat,synovial fluid, tears, vomit, urine and exhaled condensate. In someembodiments, the sample comprises a human body fluid. In someembodiments, the sample comprises at least one of cells, tissues, bodilyfluids, stool, amniotic fluid, aqueous humour, vitreous humour, blood,whole blood, fractionated blood, plasma, serum, breast milk,cerebrospinal fluid, cerumen, chyle, chime, endolymph, perilymph, feces,gastric acid, gastric juice, lymph, mucus, nasal drainage, phlegm,pericardial fluid, peritoneal fluid, pleural fluid, pus, rheum, saliva,sebum, semen, sputum, sweat, synovial fluid, tears, vomit, urine, orexhaled condensate, or a mixture thereof.

AA31. The device of any prior embodiments, wherein the fluidic samplecomprises nucleic acids or proteins, or a mixture thereof.

AA32. The device of any prior embodiments, wherein the fluidic samplecomprises DNA or RNA, or a mixture thereof.

Apparatus with Heating Source

BB1. An apparatus for rapidly changing temperature of a fluidic sample,comprising:

a holder that can hold a device of any AA embodiments; and

a heating source that is configured to supply energy to theheating/cooling layer; and

a controller that is configured to control the heating source.

BB1.1 The apparatus of any prior BB embodiments, wherein the heatingsource is configured to radiate electromagnetic waves in a range ofwavelength that the heating/cooling layer has an absorption coefficientof 50% or higher.

BB2. The apparatus of any prior BB embodiments, wherein the heatingsource comprises one or an array of light-emitting diodes (LEDs), one oran array of lasers, one or an array of lamps, or a combination ofthereof.

BB2.1. The apparatus of any prior BB embodiments, wherein the heatingsource comprises halogen lamp, halogen lamp with reflector, LED withfocusing lens, laser with focusing lens, halogen lamp with couplingoptical fiber, LED with coupling optical fiber, laser with couplingoptical fiber.

BB3. The apparatus of any prior BB embodiments, wherein the wavelengthis 50 nm, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450nm, 500 nm, 550 nm, 600 nm, 650 nm, 700 nm, 750 nm, 800 nm, 850 nm, 900nm, 950 nm, 1 um, 10 um, 25 um, 50 um, 75 um, or 100 um, or in a rangebetween any of the two values.

BB3.1 The apparatus of any prior BB embodiments, wherein the wavelengthof the electromagnetic waves is 100 nm to 300 nm, 400 nm to 700 nm(visible range), 700 nm to 1,000 nm (IR range), 1 um to 10 um, 10 um to100 um, or in a range between any of the two values.

BB4. The apparatus of any prior BB embodiments, further comprising aheat sink that is configured to absorb at least part of the heatradiated from the sample holder and/or the heating source.

BB4.1. The apparatus of any prior BB embodiments, wherein the heat sinkis chamber that at least partially encloses the device.

BB4.2. The apparatus of any prior BB embodiments, wherein the chambercomprises a lower aperture configured to allow passage ofelectromagnetic waves from the heating source to the heating/coolinglayer, and an upper aperture configured to allow imaging of the sample.

BB5. The apparatus of any prior BB embodiments, wherein the sampleholder is heated optically, electrically, by RF, or a combination ofthereof.

BB6. An apparatus for rapidly changing temperature of a fluidic sample,comprising:

a device of any AA embodiments; and

a heat sink that is configured to absorb at least part of the heatradiated from the sample holder and/or the heating source.

BB7. The apparatus of any prior BB embodiments, wherein the heat sink isa chamber that at least partially encloses the device, wherein thechamber comprises a radiation aperture configured to allow passage ofelectromagnetic waves from a heating source to the heating/coolinglayer, and an optical aperture configured to allow imaging of thesample.

BB8. The apparatus of any prior BB embodiments, further comprising acooling member attached to the chamber, wherein the cooling member isconfigured to reduce temperature in the chamber.

BB9. The apparatus of embodiment BB7, wherein the cooling member is afan.

BB10. The apparatus of embodiment BB7, wherein the cooling member is aPeltier cooler.

BB11. The apparatus of any BB embodiments, wherein the chamber has anon-reflective inner surface.

BB11.1 The apparatus of any BB embodiments, wherein the chamber has aninner surface made of black metal.

BB12. The apparatus of any BB embodiments, wherein the device issuspended (i.e. has minimum) thermal conduction contact with the chamberwall.

BB13. The apparatus of any BB embodiments, wherein the heat sink isconfigured to connect the sample holder to a mobile device.

BB13.1 The apparatus of embodiment B13, wherein the mobile device is asmartphone comprising a camera.

BB14. The apparatus of any BB embodiments, wherein the heat sinkcomprises optical elements that optimizes capturing images of the samplein the sample card.

CC1. A system for rapidly changing temperature of a fluidic sample,comprising: a device of any AA embodiments or an apparatus of any BBembodiments; and a signal sensor that is configured to senses a signalfrom the sample on the device.

CC2. The system of any prior CC embodiments, wherein the signal sensoris an optical sensor that is configured to image the fluidic sample.

CC2.1 The system of any prior CC embodiments, wherein the optical sensoris a photodetector, camera, or a device capable of capturing images ofthe fluidic sample.

CC3. The system of any prior CC embodiments, wherein the signal sensoris an electrical sensor that is configured to detect electrical signalsfrom the device.

CC4. The system of any prior CC embodiments, wherein the signal sensoris a mechanical sensor that is configured to detect mechanical signalsfrom the device.

CC5. The system of any prior CC embodiments, wherein the signal sensoris configured to monitor the amount of an analyte in the sample.

CC6. The system of any prior CC embodiments, wherein signal sensor isoutside the chamber and receive optical signals from the sample throughan optical aperture on the chamber.

CC7. The system of any CC embodiment, further comprising a thermalcoupler bound to the heating/cooling layer.

CC8. The system of any prior CC embodiments, further comprising athermostat that monitor the temperature of the heating/cooling layer.

CC9. The system of any prior CC embodiments, further comprising atemperature monitoring dye that is configured to facilitate monitoringthe temperature of the sample in the device.

CC9.1. The system of any prior CC embodiments, wherein the temperaturemonitoring dye is in liquid form.

CC9.2. The system of any prior CC embodiments, wherein the temperaturemonitoring dye comprises LDS 688, LDS 698, LDS 950, LD 390, LD 423, LD425, or IR 144, or a combination thereof.

DD1. The device, apparatus, or system of any prior embodiments, wherein:

there are spacers that are fixed on one of both of the plates, whereinat least one of the spacers is in the sample contact area;

the sample layer has a thickness of 0.1-200 um;

the first plate is in contact with the heating/cooling layer and has athickness of 500 um or less, and the second pate is not in contact withthe heating/cooling layer and has a thickness of 5 mm or less; and

the heating/cooling layer: (1) has a thickness of less than 1 mm, (2)has an area of less than 100 mm² that is substantially less than thearea of either the first or the second plate, and (3) is configured toconvert energy from electromagnetic waves into heat to raise thetemperature of at least part of the fluidic sample in the layer ofuniform thickness.

DD2. The device, apparatus, or system of any prior embodiments, wherein:

the heating/cooling layer is on the inner surface of the first plate andin contact with the sample when the plates are in the closedconfiguration;

the heating/cooling layer is made from silicon; and

there is a chamber that encloses the sample holder and the chamber has anon-reflective inner surface.

DD3. The device, apparatus, or system of any prior embodiments, wherein:

there is a heating source that is configured to radiate electromagneticwaves in a range of wavelength that the heating/cooling layer has anabsorption coefficient of 50% or higher;

there is a chamber that comprises a lower aperture configured to allowpassage of electromagnetic waves from the heating source to theheating/cooling layer, and an upper aperture configured to allow imagingof the sample; and

there is an optical sensor that is configured to capture images of thefluidic sample in the sample holder.

EE1.1. A method for rapidly changing temperature of a fluidic sample,comprising:

providing a device that comprises a first plate, a second plate, aheating layer, and a cooling layer, wherein:

each of the plates comprises, on its respective inner surface, a samplecontact area;

the heating layer is positioned on the inner surface, the outer surface,or inside of one of the plates; and is configured to heat a relevantvolume of the sample, wherein the relevant volume of the sample is aportion or an entirety of the sample that is being heated to a desiredtemperature; and

the cooling layer is positioned on the inner surface, the outer surface,or inside of one of the plates; is configured to cool the relevantsample volume; and comprises a layer of material that that has a thermalconductivity to thermal capacity ratio of 0.6 cm²/sec or larger, whereinthe high thermal conductivity to thermal capacity ratio layer has anarea larger than the lateral area of the sample volume;

wherein the distance between the cooling layer and a surface of therelevant sample volume is zero or less than a distance that isconfigured to make the thermal conductance per unit area between thecooling layer and the surface of the relevant sample volume equal to 150W/(m²·K) or larger.

depositing a fluidic sample on one or both of the sample contact areasof the respective plates;

pressing the plates, by hand, to make the sample contact areas face eachother, wherein the plates are separated by an average separationdistance of 200 um or less, sandwiching the sample between them andpressing at least part of the sample into a thin layer:

changing and/or maintaining the temperature of the relevant volume inthe device.

EE1.2. A method for rapidly changing temperature of a fluidic sample,comprising:

providing the device of the SC-A embodiments:

depositing a fluidic sample on one or both of the sample contact areasof the respective plates;

pressing the plates, by hand, to sandwich the sample between them andpressing at least part of the sample into a thin layer:

changing and/or maintaining the temperature of the relevant volume inthe device.

EE1.3. A method for rapidly changing temperature of a fluidic sample,comprising: obtaining the system of the CC embodiments;

depositing the fluidic sample in the sample holder;

pressing the first plate and the second plate to compress at least partof the sample into a layer of uniform thickness; and

changing and maintaining the temperature of the sample layer by changingthe presence, intensity, wavelength, frequency, and/or angle of theelectromagnetic waves from the heating source.

EE2. The method of any prior EE embodiments, wherein changing thetemperature of the sample layer comprises raising the temperature orlowering the temperature.

EE3. The method of any prior EE embodiments, further comprising imagingthe sample layer with the optical sensor.

EE4. The method of any prior EE embodiments, further comprisingmonitoring the temperature of the sample layer and adjusting the step ofchanging and maintaining the temperature of the sample layer.

EE5. The method of any prior EE embodiments, wherein the step ofchanging and maintaining the temperature of the sample layer isconducted according to a pre-determined program.

EE6. The method of any prior EE embodiments, wherein the method iscustomized to facilitate polymerase chain reaction (PCR) assays forchanging temperature of the sample according to a predetermined program

EE7. The method of any prior EE embodiments, further comprisingmonitoring the amount of an analyte in the sample in real time.

FF1. The device, apparatus, system or method of any prior embodiments,wherein the sample comprises nucleic acids.

FF1.1 The device, apparatus, system or method of any prior embodiments,wherein the sample comprises DNA.

FF1.2 The device, apparatus, system or method of any prior embodiments,wherein the sample comprises RNA.

FF1.3 The device, apparatus, system or method of any prior embodiments,wherein the sample comprises DNA or RNA molecule, or a DNA/RNA hybrid,or mixtures of DNA and/or RNA.

FF1.4 The device, apparatus, system or method of any prior embodiments,wherein the sample comprises genomic or chromosomal DNA, plasmid DNA,amplified DNA, cDNA, total RNA, mRNA and small RNA.

FF1.5 The device, apparatus, system or method of any prior embodiments,wherein the sample comprises natural DNA and/or RNA molecule, orsynthetic DNA and/or RNA molecule.

FF1.6 The device, apparatus, system or method of any prior embodiments,wherein the sample comprises cell-free nucleic acids, wherein“cell-free” refers to nucleic acids are not contained in any cellularstructures.

FF1.7 The device, apparatus, system or method of any prior embodiments,wherein the sample comprises nucleic acids are contained within cellularstructures, which include but not limited to human cells, animal cells,plant cells, bacterial cells, fungi cells, and/or viral particles.

FF1.8 The device, apparatus, system or method of any prior embodiments,wherein the sample comprises purified nucleic acids.

FF2. The device, apparatus, system or method of any prior embodiments,wherein the sample comprises proteins and/or lipids.

FF3. The device, apparatus, system or method of any prior embodiments,wherein the sample comprises reagents configured for nucleic acidamplification.

FF3.1. The device, apparatus, system or method of any prior embodiments,wherein the sample comprises a pre-mixed polymerase chain reaction (PCR)medium.

FF3.2. The device, apparatus, system or method of any prior embodiments,wherein the sample comprises reagents configured to detect nucleic acidsby amplifying (generating numerous copies of) the target molecules insamples, wherein target molecule refers to a sequence, or partialsequence, of nucleic acid of interest.

FF3.3. The device, apparatus, system or method of any prior embodiments,wherein the nucleic acid amplification refers to nucleic acidamplification techniques include but not limited to, differentpolymerase chain reaction (PCR) methods, such as hot-start PCR, nestedPCR, touchdown PCR, reverse transcription PCR, RACE PCR, digital PCR,etc., and isothermal amplification methods, such as Loop-mediatedisothermal amplification (LAMP), strand displacement amplification,helicase-dependent amplification, nicking enzyme amplification, rollingcircle amplification, recombinase polymerase amplification, etc.

FF3.4. The device, apparatus, system or method of any prior embodiments,wherein the reagents comprise primers, deoxynucleotides (dNTPs),bivalent cations (e.g. Mg2+), monovalent cation (e.g. K+), buffersolutions, enzymes, or reporters, or any combination or mixture thereof.

FF3.5. The device, apparatus, system or method of any prior embodiments,wherein the reagents are either in the dry form on the inner surface ofthe first or the second plate or both, or in a liquid form encased in,embedded in, or surrounded by, a material that melts with increasingtemperatures, such as, for example, paraffin.

FF3.6. The device, apparatus, system or method of any prior embodiments,wherein primers comprise one or more pairs of forward and reverseprimers.

FF3.7. The device, apparatus, system or method of any prior embodiments,wherein the reagents comprise DNA-dependent polymerase, or RNA-dependentDNA polymerase, or DNA-dependent RNA polymerase.

FF3.8. The device, apparatus, system or method of any prior embodiments,wherein the reagents comprise “reporters” that refer to any tag, label,or dye that can bind to, or intercalate within, the nucleic acidmolecule or be activated by byproducts of the amplification process toenable visualization of the nucleic acid molecule or the amplificationprocess.

FF3.8.1 The device, apparatus, system or method of any priorembodiments, wherein the reports include but are not limited tofluorescent labels or tags or dyes, intercalating agents, molecularbeacon labels, or bioluminescent molecules, or a combination thereof.

FF3.9. The device, apparatus, system or method of any prior embodiments,wherein the reagents comprise cell lysing reagent, which is configuredto facilitate breaking down cellular structures.

FF3.9.1. The device, apparatus, system or method of any priorembodiments, wherein the cell lysing reagent includes but not limited tosalts, detergents, enzymes, and other additives.

FF3.9.2. The device, apparatus, system or method of any priorembodiments, wherein the salt includes but not limited to lithium salt(e.g. lithium chloride), sodium salt (e.g. sodium chloride), potassium(e.g. potassium chloride).

FF3.9.2. The device, apparatus, system or method of any priorembodiments, wherein the detergents are ionic, including anionic andcationic, non-ionic or zwitterionic.

FF3.9.3. The device, apparatus, system or method of any priorembodiments, wherein the ionic detergent includes any detergent which ispartly or wholly in ionic form when dissolved in water.

FF3.9.4. The device, apparatus, system or method of any priorembodiments, wherein anionic detergents include but not limited tosodium dodecyl sulphate (SDS) or other alkali metal alkylsulphate saltsor similar detergents, sarkosyl, or combinations thereof.

FF3.10. The device, apparatus, system or method of any priorembodiments, wherein enzymes includes but not limited to lysozyme,cellulose, and proteinase.

FF3.11. The device, apparatus, system or method of any priorembodiments, wherein chelating agents include but not limited to EDTA,EGTA and other polyamino carboxylic acids, and some reducing agents,such as dithiotreitol (dTT).

FF4. The device, apparatus, system or method of any prior embodiments,wherein the sample comprises an analyte the amount of which is changedwith the temperature changes.

FF5. The device, apparatus, system or method of any prior embodiments,wherein the sample comprises human bodily fluids, such as but notlimited to whole blood, plasma, serum, urine, saliva, and sweat, andcell cultures (mammalian, plant, bacteria, fungi), and a combination ormixture thereof.

FF6. The device, apparatus, system or method of any prior embodiments,wherein the sample is freshly obtained, stored or treated in any desiredor convenient way, for example by dilution or adding buffers, or othersolutions or solvents.

FF7. The device, apparatus, system or method of any prior embodiments,wherein the sample comprises cellular structures such as but not limitedto human cells, animal cells, plant cells, bacteria cells, fungus cells,and virus particles, and a combination or mixture thereof.

GG1. The device, apparatus, system or method of any prior embodiments,wherein an analyte in the sample is stained.

GG2. The device, apparatus, system or method of any prior GGembodiments, wherein the amount of the analyte is measured byfluorescence intensity.

GG3. The device, apparatus, system or method of any prior GGembodiments, wherein the amount of the analyte is measured bycolorimetric intensity.

GG4. The device, apparatus, system or method of any prior embodiments,wherein the analyte is nucleic acid, which is stained with ethidiumbromide (EB), methylene blue,

SYBR green I, SYBR green II, pyronin Y, DAPI, acridine orange, orNancy-520, or a combination thereof.

GG5. The device, apparatus, system or method of any prior embodiments,wherein the analyte is DNA, which is stained with ethidium bromide (EB),methylene blue, pyronin Y, DAPI, acridine orange, or Nancy-520, or acombination thereof, and measured with fluorescence intensity.

GG6. The device, apparatus, system or method of any prior embodiments,wherein the analyte is DNA, which is stained with ethidium bromide (EB),methylene blue, pyronin Y, DAPI, acridine orange, or Nancy-520, or acombination thereof, and measured with colorimetric intensity.

GG7. The device, apparatus, system or method of any prior embodiments,wherein the analyte is RNA, which is stained with ethidium bromide (EB),methylene blue, SYBR green II, pyronin Y, or acridine orange, or acombination thereof, and measured with fluorescence intensity.

GG8. The device, apparatus, system or method of any prior embodiments,wherein the analyte is RNA, which is stained with ethidium bromide (EB),methylene blue, SYBR green II, pyronin Y, or acridine orange, or acombination thereof, and measured with colorimetric intensity.

GG9. The device, apparatus, system or method of any prior embodiments,wherein the analyte is nucleic acid to be detected by reporters.

GG9.1. The device, apparatus, system or method of any prior embodiments,wherein the reporters include but not limited to tag, label, or dye thatcan bind to, or intercalate within, the nucleic acid molecule or beactivated by byproducts of the amplification process to enablevisualization of the nucleic acid molecule or the amplification process.

GG9.2. The device, apparatus, system or method of any prior embodiments,wherein the reporters include but are not limited to fluorescent labelsor tags or dyes, intercalating agents, molecular beacon labels, orbioluminescent molecules, or a combination thereof.

GG9.3. The device, apparatus, system or method of any prior embodiments,wherein the amount of reporter is measured by colorimetric intensityand/or by fluorescence intensity.

HH1. The device, apparatus, system or method of any prior embodiments,wherein the device, apparatus, system or method is configured tofacilitate PCR assays for changing temperature of the sample accordingto a predetermined program.

HH2. The device, apparatus, system or method of any prior embodiments,wherein the device, apparatus, system or method is configured to conductdiagnostic testing, health monitoring, environmental testing, and/orforensic testing.

HH3. The device, apparatus, system or method of any prior embodiments,wherein the device, apparatus, system or method is configured to conductDNA amplification, DNA quantification, selective DNA isolation, geneticanalysis, tissue typing, oncogene identification, infectious diseasetesting, genetic fingerprinting, and/or paternity testing.

HH4. The device, apparatus, system or method of any prior embodiments,wherein the device, apparatus, system or method is configured to conductreal time PCR.

HH5. The device, apparatus, system or method of any prior embodiments,wherein the device, apparatus, system or method is configured to conductnucleic acid amplification.

HH5.1 The device, apparatus, system or method of any prior embodiments,wherein nucleic acid amplification includes any techniques used todetect nucleic acids by amplifying (generating numerous copies of) thetarget molecules in samples, wherein target molecule refers to asequence, or partial sequence, of nucleic acid of interest.

HH6 The device, apparatus, system or method of any prior embodiments,wherein the device, apparatus, system or method is configured to conductnucleic acid amplification techniques include but not limited to,different polymerase chain reaction (PCR) methods, such as hot-startPCR, nested PCR, touchdown PCR, reverse transcription PCR, RACE PCR,digital PCR, etc., and isothermal amplification methods, such asLoop-mediated isothermal amplification (LAMP), strand displacementamplification, helicase-dependent amplification, nicking enzymeamplification, rolling circle amplification, recombinase polymeraseamplification, etc.

A1. A device for rapidly changing temperature of a thin fluidic samplelayer, comprising:

a first plate, a second plate, and a heating/cooling layer, wherein:

the heating/cooling layer is on one of the plates,

each of the plates comprises, on its respective surface, a samplecontact area for contacting a fluidic sample; and

the plates have a configuration for rapidly changing temperature of thesample, in which:

the sample contact areas face each other and are significant parallel,

the average spacing between the contact areas is equal to or less than200 microns,

the two plates regulate (or confine) at least part of the sample into alayer of highly uniform thickness and substantially stagnant relative tothe plates,

the heating/cooling layer is near the at least part of the sample ofuniform thickness,

the area of the at least part of the sample and the heating/coolinglayer are substantially larger than the uniform thickness.

A2. The device of embodiment A1, wherein the heating/cooling layercomprises a disk-coupled dots-on-pillar antenna (D2PA) array, siliconsandwich, graphene, back materials, superlattice or other plasmonicmaterials, other a combination thereof.

A3. The device of embodiment A1, wherein the heating/cooling layercomprises carbon or black nanostructures or a combination thereof.

A4. The device of any of embodiments A1-A3, wherein the heating/coolinglayer is configured to absorb radiation energy.

A5. The device of any of embodiments A1-A4, wherein the heating/coolinglayer is configured to radiate energy in the form of heat afterabsorbing radiation energy.

A6. The device of any of embodiments A1-A5, wherein the heating/coolinglayer is positioned underneath the sample layer and in direct contactwith the sample layer.

A7. The device of any of embodiments A1-A6, wherein the heating/coolinglayer is configured to absorbing electromagnetic waves selected from thegroup consisting of: radio waves, microwaves, infrared waves, visiblelight, ultraviolet waves, X-rays, gamma rays, and thermal radiation.

A8. The device of any of embodiments A1-A7, wherein at least one of theplates does not block the radiation that the heating/cooling layerabsorbs.

A9. The device of any of embodiments A1-A8, wherein one or both of theplates have low thermal conductivity.

A10. The device of any of embodiments A1-A9, wherein the uniformthickness of the sample layer is regulated by one or more spacers thatare fixed to one or both of the plates.

A11. The device of any of embodiments A1-A10, wherein the sample is apre-mixed polymerase chain reaction (PCR) medium.

A12. The device of embodiment A11, wherein the device is configured tofacilitate PCR assays for changing temperature of the sample accordingto a predetermined program.

A13. The device of any of embodiments A1-A12, wherein the device isconfigured to conduct diagnostic testing, health monitoring,environmental testing, and/or forensic testing.

A14. The device of any of embodiments A1-A13, wherein the device isconfigured to conduct DNA amplification, DNA quantification, selectiveDNA isolation, genetic analysis, tissue typing, oncogene identification,infectious disease testing, genetic fingerprinting, and/or paternitytesting.

A15. The device of any of embodiment A1-A14, wherein the sample layer islaterally sealed to reduce sample evaporation.

B1. A system for rapidly changing temperature of a thin fluidic samplelayer, comprising:

a first plate, a second plate, a heating/cooling layer, and a heatingsource, wherein:

the heating/cooling layer is on one of the plates;

the heating source is configured to radiate electromagnetic waves thatthe heating/cooling layer absorbs significantly;

each of the plates comprises, on its respective surface, a samplecontact area for contacting a fluidic sample; and

the plates have a configuration for rapidly changing temperature of thesample, in which:

the sample contact areas face each other and are significant parallel,

the average spacing between the contact areas is equal to or less than200 μm,

the two plates confine at least part of the sample into a layer ofhighly uniform thickness and substantially stagnant relative to theplates,

the heating/cooling layer is near the at least part of the sample ofuniform thickness,

the area of the at least part of the sample and the heating/coolinglayer are substantially larger than the uniform thickness.

B2. The system of embodiment B1, wherein the heating/cooling layercomprises a disk-coupled dots-on-pillar antenna (D2PA) array, siliconsandwich, graphene, superlattice or other plasmonic materials, other acombination thereof.

B3. The system of embodiment B1, wherein the heating/cooling layercomprises carbon or black nanostructures or a combination thereof.

B4. The system of any of embodiments B1-B3, wherein the heating/coolinglayer is configured to absorb at least 80% of the radiation energy fromthe electromagnetic waves from the heating source.

B5. The system of any of embodiments B1-B4, wherein the heating/coolinglayer is configured to radiate energy in the form of heat afterabsorbing radiation energy.

B6. The system of any of embodiments B1-B5, wherein the heating/coolinglayer is positioned underneath the sample layer and in direct contactwith the sample layer.

B7. The system of any of embodiments B1-B6, wherein the heating/coolinglayer is configured to absorbing electromagnetic waves selected from thegroup consisting of: radio waves, microwaves, infrared waves, visiblelight, ultraviolet waves, X-rays, gamma rays, and thermal radiation.

B8. The system of any of embodiments B1-B7, wherein at least one of theplates does not block the radiation from the heating source.

B9. The system of any of embodiments B1-B8, wherein one or both of theplates have low thermal conductivity.

B10. The system of any of embodiments B1-B9, wherein the uniformthickness of the sample layer is regulated by one or more spacers thatare fixed to one or both of the plates.

B11. The system of any of embodiments B1-B10, wherein the sample is apre-mixed polymerase chain reaction (PCR) medium.

B12. The system of embodiment B11, wherein the system is configured tofacilitate PCR assays for changing temperature of the sample accordingto a predetermined program.

B13. The system of any of embodiments B1-B12, wherein the system isconfigured to conduct diagnostic testing, health monitoring,environmental testing, and/or forensic testing.

B14. The system of any of embodiments B1-B15, wherein the system isconfigured to conduct DNB amplification, DNB quantification, selectiveDNB isolation, genetic analysis, tissue typing, oncogene identification,infectious disease testing, genetic fingerprinting, and/or paternitytesting.

B15. The system of any of embodiments B1-B14, wherein the sample layeris laterally sealed to reduce sample evaporation.

B16. The system of any of embodiments B1-B15, further comprising acontroller, which is configured to control the presence, intensity,wavelength, frequency, and/or angle of the electromagnetic waves.

B17. The system of any of embodiments B1-B16, further comprising athermometer, which is configured to measure the temperature at or inproximity of the sample contact area and send a signal to the controllerbased on the measured temperature.

B18. The system of embodiment B17, wherein the thermometer is selectedfrom the group consisting of: fiber optical thermometer, infraredthermometer, liquid crystal thermometer, pyrometer, quartz thermometer,silicon bandgap temperature sensor, temperature strip, thermistor, andthermocouple.

C1. A system for facilitating a polymerase chain reaction (PCR) byrapidly changing temperature of a thin fluidic PCR sample layer,comprising:

a first plate, a second plate, a heating/cooling layer, a heatingsource, and a controller wherein:

the heating/cooling layer is on one of the plates;

the heating source is configured to radiate electromagnetic waves thatthe heating/cooling layer absorbs significantly;

each of the plates comprises, on its respective surface, a samplecontact area for contacting a fluid PCR sample, which is a pre-mixed PCRmedium;

the controller is configured to control the heating source and rapidlychange the temperature of the sample according to a predeterminedprogram; and

the plates have a configuration for rapidly changing temperature of thesample, in which:

the sample contact areas face each other and are significant parallel,

the average spacing between the contact areas is equal to or less than200 μm,

the two plates confine at least part of the sample into a layer ofhighly uniform thickness and substantially stagnant relative to theplates,

the heating/cooling layer is near the at least part of the sample ofuniform thickness, and

the area of the at least part of the sample and the heating/coolinglayer are substantially larger than the uniform thickness.

C2. The system of embodiment C1, wherein the controller is configured tocontrol the present, intensity, wavelength, frequency, and/or angle ofthe electromagnetic waves from the heating source.

C3. The system of embodiment C1 or C2, wherein the heating source andthe heating/cooling layer are configured that the electromagnetic wavescause an average ascending temperature rate ramp of at least 10° C./s;and the removal of the electromagnetic waves results in an averagedescending temperature rate ramp of at least 5° C./s.

C4. The system of any of embodiments C1-C2, wherein the heating sourceand the heating/cooling layer are configured to create an averageascending temperature rate ramp of at least 10° C./s and an averagedescending temperature rate ramp of at least 5° C./s.

C5. The system of any of embodiments C1-C2, wherein the heating sourceand the heating/cooling layer are configured to create an averageascending temperature rate ramp of at least 10° C./s to reach theinitialization step, the denaturation step and/or theextension/elongation step during a PCR, and an average descendingtemperature rate ramp of at least 5° C./s to reach the annealing stepand/or the final cooling step during a PCR.

C6. The system of any of embodiments C1-C5, wherein the PCR samplecomprises: template DNA, primer DNA, cations, polymerase, and buffer.

D1. A method for rapidly changing temperature of a thin fluidic samplelayer, comprising:

providing a first plate a second plate, each of the plates comprising,on its respective inner surface, a sample contact area;

providing a heating/cooling layer and a heating source, wherein theheating/cooling layer is on one of the plates, and the heating source isconfigured to radiate electromagnetic waves that the heating/coolinglayer absorbs significantly;

depositing a fluidic sample on one or both of the plates;

pressing the plates into a closed configuration, in which:

the sample contact areas face each other and are significant parallel,

the average spacing between the contact areas is equal to or less than200 μm,

the two plates confine at least part of the sample into a layer ofhighly uniform thickness and substantially stagnant relative to theplates;

the heating/cooling layer is near the at least part of the sample ofuniform thickness,

the area of the at least part of the sample and the heating/coolinglayer are substantially larger than the uniform thickness; and

changing and maintaining the temperature of the sample layer by changingthe presence, intensity, wavelength, frequency, and/or angle of theelectromagnetic waves from the heating source.

D2. The method of embodiment D1, wherein the step of pressing the platesinto a closed figuration comprises pressing the plates with an imprecisepressing force.

D3. The method of embodiment D1 or D2, wherein the step of pressing theplates into a closed figuration comprises pressing the plates directedlywith human hands.

D4. The method of any of embodiments D1-D3, wherein the layer of highlyuniform thickness has a thickness variation of less than 10%.

D5. The method of any of embodiments D1-D4, wherein the heating/coolinglayer comprises a disk-coupled dots-on-pillar antenna (D2PA) array,silicon sandwich, graphene, superlattice or other plasmonic materials,other a combination thereof.

D6. The method of any of embodiments D1-D5, wherein the heating/coolinglayer comprises carbon or black nanostructures or a combination thereof.

D7. The method of any of embodiments D1-D6, wherein the heating/coolinglayer is configured to absorb at least 80% of the radiation energy fromthe electromagnetic waves from the heating source.

D8. The method of any of embodiments D1-D7, wherein the heating/coolinglayer is configured to radiate energy in the form of heat afterabsorbing radiation energy.

D9. The method of any of embodiments D1-D8, wherein the heating/coolinglayer is positioned underneath the sample layer and in direct contactwith the sample layer.

D10. The method of any of embodiments D1-D9, wherein the heating/coolinglayer is configured to absorbing electromagnetic waves selected from thegroup consisting of: radio waves, microwaves, infrared waves, visiblelight, ultraviolet waves, X-rays, gamma rays, and thermal radiation.

D11. The method of any of embodiments D1-D10, wherein at least one ofthe plates does not block the radiation from the heating source.

D12. The method of any of embodiments D1-D11, wherein one or both of theplates have low thermal conductivity.

D13. The method of any of embodiments D1-D12, wherein the uniformthickness of the sample layer is regulated by one or more spacers thatare fixed to one or both of the plates.

D14. The method of any of embodiments D1-D13, wherein the sample is apre-mixed polymerase chain reaction (PCR) medium.

D15. The method of embodiment D14, wherein the method is used tofacilitate PCR assays for changing temperature of the sample accordingto a predetermined program.

D16. The method of any of embodiments D1-D15, wherein the method is usedto conduct diagnostic testing, health monitoring, environmental testing,and/or forensic testing.

D17. The method of any of embodiments D1-D16, wherein the method is usedto conduct DNB amplification, DNB quantification, selective DNBisolation, genetic analysis, tissue typing, oncogene identification,infectious disease testing, genetic fingerprinting, and/or paternitytesting.

D18. The method of any of embodiments D1-D17, wherein the sample layeris laterally sealed to reduce sample evaporation.

D19. The method of any of embodiments D1-D18, wherein the heating sourceis controlled by a controller, which is configured to control thepresence, intensity, wavelength, frequency, and/or angle of theelectromagnetic waves.

D20. The method of any of embodiments D1-D19, wherein the controller isconfigured to receive signals from a thermometer, which is configured tomeasure the temperature at or in proximity of the sample contact areaand send a signal to the controller based on the measured temperature.

D21. The method of embodiment D20, wherein the thermometer is selectedfrom the group consisting of: fiber optical thermometer, infraredthermometer, liquid crystal thermometer, pyrometer, quartz thermometer,silicon bandgap temperature sensor, temperature strip, thermistor, andthermocouple.

E1. A method for facilitating a polymerase chain reaction (PCR) byrapidly changing temperatures in a fluidic PCR sample, comprising:

providing a first plate a second plate, each of the plates comprising,on its respective inner surface, a sample contact area;

providing a heating/cooling layer, a heating source and a controller,wherein the heating/cooling layer is on one of the plates, and theheating source is configured to radiate electromagnetic waves that theheating/cooling layer absorbs significantly;

depositing a fluidic PCR sample on one or both of the plates;

pressing the plates into a closed configuration, in which:

the sample contact areas face each other and are significant parallel,

the average spacing between the contact areas is equal to or less than200 μm,

the two plates confine at least part of the PCR sample into a layer ofhighly uniform thickness and substantially stagnant relative to theplates;

the heating/cooling layer is near the at least part of the PCR sample ofuniform thickness,

the area of the at least part of the sample and the heating/coolinglayer are substantially larger than the uniform thickness; and

using the controller to control the heating source to conduct a PCR bychanging and maintaining the temperature of the PCR sample layeraccording to a predetermined program, wherein when the temperatures arechanged, the heating source creates an average ascending temperaturerate ramp of at least 10° C./s and an average descending temperaturerate ramp of at least 5° C./s during the PCR.

E2. The method of embodiment E1, wherein changing and maintaining thetemperature of the PCR sample layer is achieved by adjusting theintensity, wavelength, frequency, and/or angle of the electromagneticwaves from the heating source.

E3. The system of any of embodiments E1-E2, wherein the heating sourceand the heating/cooling layer are configured to create an averageascending temperature rate ramp of at least 10° C./s to reach theinitialization step, the denaturation step and/or theextension/elongation step during a PCR, and an average descendingtemperature rate ramp of at least 5° C./s to reach the annealing stepand/or the final cooling step during a PCR.

E4. The method of any of embodiments E1-E3, wherein the PCR samplecomprises: template DNA, primer DNA, cations, polymerase, and buffer.

NN1 A device for rapidly changing temperature of a thin fluidic samplelayer, comprising:

a first plate, and a second plate, wherein:

each of the plates comprises, on its respective surface, a samplecontact area for contacting a fluidic sample; and

the plates have a configuration for rapidly changing temperature of thesample, in which:

the sample contact areas face each other and are significant parallel,

the average spacing between the contact areas is equal to or less than200 microns,

the two plates regulate (or confine) at least part of the sample into alayer of highly uniform thickness and substantially stagnant relative tothe plates,

the heating/cooling layer is near the at least part of the sample ofuniform thickness,

the area of the at least part of the sample and the heating/coolinglayer are substantially larger than the uniform thickness.

JJ1. The device of any prior embodiments, further comprising a hingethat connects the first plate and the second plate, and is configured toallow the two plates to rotate around the hinge into differentconfigurations.

JJ2. The device of any prior embodiments, wherein after relativeposition of the plates are adjusted by an external force, the hingemaintains an angle between the two plates that is within 5 degrees fromthe angle just before the external force is removed.

JJ3. The device of any prior embodiments, wherein the wherein afterrelative position of the plates are adjusted by an external force, thehinge maintains an angle between the two plates that is within 10degrees from the angle just before the external force is removed.

JJ4. The device of any prior embodiments, wherein the hinge is made of apiece of a piece of hinge material of a substantially uniform thickness,wherein the hinge material is attached to a part of the inner surface ofthe first plate and a part of the outer surface of the second plate, andthe attachments do not completely separate using operation.

JJ5. The device of any prior embodiments, wherein the hinge is made of apiece of hinge material of a substantially uniform thickness, whereinthe hinge material is attached a part of the outer surfaces of the firstplate and the second plate, and the attachments do not completelyseparate using operation.

JJ6. The device of any prior embodiments, wherein the hinge material isa metal.

JJ7. The device of any prior embodiments, wherein the hinge materials isselected from a group consisting of: gold, silver, copper, aluminum,iron, tin, platinum, nickel, cobalt, and alloys thereof.

JJ8. The device of any prior embodiments, wherein the hinge comprises afirst leaf, a second leaf, and a joint that connects the leaves and isconfigured for the leaves to rotate around the joint,

wherein the first leaf is attached to the first plate inner surfacewithout wrapping around any edge of the first plate, the second leaf isattached to the second plate outer surface, and the joint is positionedlongitudinally parallel to the hinge edge of the second plate, allowingthe two plates to rotate around the joint

KK1. The device of any prior embodiments, wherein:

one of the plate comprises one or more open notches on an edge orcorners of the plate, and

at or near the closed configuration, an edge of the other plate isconfigured to overlap with the open notch

KK2. The device of any prior embodiments, wherein the notch facilitateschanging the plates from a configuration that is near or at closedconfiguration to an open configuration for sample deposition.

KK3. The device of any prior embodiments, wherein the width of at leastone notch is in the range of ⅙ to ⅔ of the width of the notched edge.

KK4. The device of any prior embodiments, wherein the opening edge ofthe plate without the notch is inside the notched edge except for thepart over the notch.

KK5. The device of any prior embodiments, wherein the first platecomprises one or more notched edges, each of which has at least onenotch; and the second plate comprises one or more corresponding openingedges juxtaposed over the notches, allowing a user to push against oneof the opening edges over the notch to switch the two plates between theclosed configuration and the open configuration or to change the angleformed by the first plate and the second plate

KK6. The device of any prior embodiments, wherein the notch ispositioned at an intersection of two neighboring notched edges.

LL1. The device of any prior embodiments, wherein any prior deviceembodiment, wherein each of the plate further comprises, on itsrespective outer surface, a force area for applying a pressing forcethat forces the plates together, and wherein the force is an impreciseforce that has a magnitude which is, at the time that the force isapplied, either (a) unknown and unpredictable, or (b) cannot be knownand cannot be predicted within an accuracy equal or better than 30% ofthe force applied.

LL2. The device of any prior embodiments, wherein each of the platefurther comprises, on its respective outer surface, a force area forapplying a pressing force that forces the plates together, and whereinthe force is an imprecise force that has a magnitude which cannot, atthe time that the force is applied, be determined within an accuracyequal or better than 30%, 40%, 50%, 70%, 100%, 200%, 300%, 500%, 1000%,2000%, or any range between the two values.

LL3. The device of any prior embodiments, wherein the imprecise force isprovided by human hand.

MM1. The device, apparatus, system, or method of any prior embodiments,wherein the first plate and the second plate are flexible plastic filmand/or thin glass film, that each has a substantially uniform thicknessof a value selected from a range between 1 um to 25 um.

MM2. The device, apparatus, system, or method of any prior embodiments,wherein each plate has an area in a range of 1 cm{circumflex over ( )}2to 16 cm{circumflex over ( )}2.

MM3. The device, apparatus, system, or method of any prior embodiments,wherein the sample sandwiched between the two plate has a thickness of40 um or less.

MM4. The device, apparatus, system, or method of any prior embodiments,wherein the relevant sample to the entire sample ratio (RE ratio) is 12%or less.

MM5. The device, apparatus, system, or method of any prior embodiments,wherein the cooling zone is at least 9 times larger than the heatingzone.

MM6. The device, apparatus, system, or method of any prior embodiments,wherein the sample to non-sample thermal mass ratio is 2.2 or lager.

MM7. The device, apparatus, system, or method of any prior embodiments,wherein the RHC card does not comprise spacer.

MM8. The device, apparatus, system, or method of any prior embodiments,wherein the RHC card comprises spacers that are fixed on one or both ofthe plates.

MM9. The device, apparatus, system, or method of any prior embodiments,wherein:

the first plate and second plates are plastic or a thin glass. The firstplate and second plate have a thickness of 100 nm, 500 nm, 1 um, 5 um,10 um, in a range between any of the two values;

the sample between the two plates has a thickness of 5 um, 10 um, 30 um,50 um, 100 um, or in a range between any of the two values;

the distance from the H/C layer to the sample is 10 nm, 100 nm, 500 nm,1 um, 5 um, 10 um, or in a range between any of the two values;

the ratio of the cooling zone area to the relevant sample area is 16, 9,4, 2, or in a range between any of the two values;

the ratio of the cooling zone area to the heating area is 16, 9, 4, 2,or in a range between any of the two values; AND

the distance between the H/C layer and the heating source (e.g. LED) is5 mm, 10 mm, 20 mm, 30 mm, or in a range between any of the two values.

MM10. The device, apparatus, system, or method of any prior embodiments,wherein:

the first plate and second plates are plastic or a thin glass. The firstplate and second plate have a thickness of 100 nm, 500 nm, 1 um, 5 um,10 um, in a range between any of the two values;

the sample between the two plates has a thickness of 5 um, 10 um, 30 um,50 um, 100 um, or in a range between any of the two values;

the distance from the H/C layer to the sample is 10 nm, 100 nm, 500 nm,1 um, 5 um, 10 um, or in a range between any of the two values;

the ratio of the cooling zone area to the relevant sample area is 16, 9,4, 2, or in a range between any of the two values;

the ratio of the cooling zone area to the heating area is 16, 9, 4, 2,or in a range between any of the two values; OR

the distance between the H/C layer and the heating source (e.g. LED) is5 mm, 10 mm, 20 mm, 30 mm, or in a range between any of the two values.

MM11. The device, apparatus, system, or method of any prior embodiments,wherein:

the first plate and second plates are plastic or a thin glass. The firstplate has a thickness of 10 um, 25 um, 50 um, or in a range between anyof the two values; while the second plate (that plate that has heatinglayer or cooling layer) has a thickness of 100 nm, 500 nm, 1 um, 5 um,10 um, in a range between any of the two values;

the sample between the two plates has a thickness of 5 um, 10 um, 30 um,50 um, 100 um, or in a range between any of the two values;

the distance between the H/C layer and the sample is 10 nm, 100 nm, 500nm, 1 um, 5 um, 10 um, or in a range between any of the two values;

the ratio of the cooling zone area to the relevant sample area is 16, 9,4, 2, or in a range between any of the two values;

the ratio of the cooling zone area to the heating area is 16, 9, 4, 2,or in a range between any of the two values; AND

the distance between the H/C layer and the heating source (e.g. LED) is5 mm, 10 mm, 20 mm, 30 mm, or in a range between any of the two values.

MM12. The device, apparatus, system, or method of any prior embodiments,wherein:

the first plate and second plates are plastic or a thin glass. The firstplate has a thickness of 10 um, 25 um, 50 um, or in a range between anyof the two values; while the second plate (that plate that has heatinglayer or cooling layer) has a thickness of 100 nm, 500 nm, 1 um, 5 um,10 um, in a range between any of the two values;

the sample between the two plates has a thickness of 5 um, 10 um, 30 um,50 um, 100 um, or in a range between any of the two values;

the distance between the H/C layer and the sample is 10 nm, 100 nm, 500nm, 1 um, 5 um, 10 um, or in a range between any of the two values;

the ratio of the cooling zone area to the relevant sample area is 16, 9,4, 2, or in a range between any of the two values;

the ratio of the cooling zone area to the heating area is 16, 9, 4, 2,or in a range between any of the two values; OR

the distance between the H/C layer and the heating source (e.g. LED) is5 mm, 10 mm, 20 mm, 30 mm, or in a range between any of the two values.

MM13. The device, apparatus, system, or method of any prior embodiments,wherein:

the first plate and second plates are plastic or a thin glass. The firstplate and second plate have a thickness of 100 nm, 500 nm, 1 um, 5 um,10 um, 25 um, 50 um, 100 um, 175 um, 250 um, or in a range between anyof the two values;

the sample between the two plates has a thickness of 100 nm, 500 nm, 1um, 5 um, 10 um, 25 um, 50 um, 100 um, 250 um, or in a range between anyof the two values;

the distance between the H/C layer and the sample is 100 nm, 500 nm, 1um, 5 um, 10 um, 25 um, 50 um, 100 um, 175 um, 250 um, or in a rangebetween any of the two values;

the ratio of the cooling zone area to the relevant sample area is 100,64, 16, 9, 4, 2, 1, 0.5, 0.1, or in a range between any of the twovalues;

the ratio of the cooling zone area to the heating zone is 100, 64, 16,9, 4, 2, 1, 0.5, 0.1, or in a range between any of the two values; AND

the distance between the H/C layer and the heating source (e.g. LED) is500 um, 1 mm, 3 mm, 5 mm, 10 mm, 20 mm, 30 mm, or in a range between anyof the two values.

MM14. The device, apparatus, system, or method of any prior embodiments,wherein:

the first plate and second plates are plastic or a thin glass. The firstplate and second plate have a thickness of 100 nm, 500 nm, 1 um, 5 um,10 um, 25 um, 50 um, 100 um, 175 um, 250 um, or in a range between anyof the two values;

the sample between the two plates has a thickness of 100 nm, 500 nm, 1um, 5 um, 10 um, 25 um, 50 um, 100 um, 250 um, or in a range between anyof the two values;

the distance between the H/C layer and the sample is 100 nm, 500 nm, 1um, 5 um, 10 um, 25 um, 50 um, 100 um, 175 um, 250 um, or in a rangebetween any of the two values;

the ratio of the cooling zone area to the relevant sample area is 100,64, 16, 9, 4, 2, 1, 0.5, 0.1, or in a range between any of the twovalues;

the ratio of the cooling zone area to the heating zone is 100, 64, 16,9, 4, 2, 1, 0.5, 0.1, or in a range between any of the two values; OR

the distance between the H/C layer and the heating source (e.g. LED) is500 um, 1 mm, 3 mm, 5 mm, 10 mm, 20 mm, 30 mm, or in a range between anyof the two values.

MM15. The device, apparatus, system, or method of any prior embodiments,wherein a light pipe collimates the light from a light source (e.g. LED)into the heating zone; the light pipe comprises a structure with ahollow hole (e.g. a tube or a structure milled a hole) with a reflectivewall; and the light pipe has a lateral dimension for 1 mm to 8 mm andlength of 2 mm to 5o mm.

MM16. The device, apparatus, system, or method of any prior embodiments,wherein:

the first plate and second plates are plastic or a thin glass;

the first plate and second plate have a thickness of 100 nm, 500 nm, 1um, 5 um, 10 10 um, in a range between any of the two values;

the sample between the two plates has a thickness in a range of 1 to 5um, 5 um to um, 10 to 30 um, or 30 um to 50 um;

the distance from the H/C layer to the sample is in a range of 10 nm to100 nm, 100 nm to 500 nm, 500 nm to 1 um, 1 um to 5 um, 5 um to 10 um,or 10 um to 25 um;

the ratio of the cooling zone area to the relevant sample area is 16, 9,4, 2, or in a range between any of the two values;

the ratio of the cooling zone area to the heating area is 16, 9, 4, 2,or in a range between any of the two values;

the distance between the H/C layer and the heating source (e.g. LED) is5 mm, 10 mm, 20 mm, 30 mm, or in a range between any of the two values;

the KC ratio for the cooling layer is in a range of between 0.5 cm²/secand 0.7 cm²/sec, 0.7 cm²/sec and 0.9 cm²/sec, 0.9 cm²/sec and 1 cm²/sec,1 cm²/sec and 1.1 cm²/sec, 1.1 cm²/sec and 1.3 cm²/sec, 1.3 cm²/sec and1.6 cm²/sec, 1.6 cm²/sec and cm²/sec, or 2 cm²/sec and cm²/sec; and

the sample to non-sample thermal mass ratio is in a range of between 0.2to 0.5, 0.5 to 0.7, 0.7 to 1, 1 to 1.5, 1.5 to 5, 5 to 10, 10 to 30, 30to 50, or 50 to 100.

MM17. The device, apparatus, system, or method of any prior embodiments,wherein:

the first plate and second plates are plastic or a thin glass;

the first plate and second plate have a thickness of 100 nm, 500 nm, 1um, 5 um, 10 um, in a range between any of the two values;

the sample between the two plates has a thickness in a range of 1 to 5um, 5 um to 10 um, 10 to 30 um, or 30 um to 50 um;

the distance from the H/C layer to the sample is in a range of 10 nm to100 nm, 100 nm to 500 nm, 500 nm to 1 um, 1 um to 5 um, 5 um to 10 um,or 10 um to 25 um;

the ratio of the cooling zone area to the relevant sample area is 16, 9,4, 2, or in a range between any of the two values;

the ratio of the cooling zone area to the heating area is 16, 9, 4, 2,or in a range between any of the two values;

the distance between the H/C layer and the heating source (e.g. LED) is5 mm, 10 mm, 20 mm, 30 mm, or in a range between any of the two values;

the KC ratio for the cooling layer is in a range of between 0.5 cm²/secand 0.7 cm²/sec, 0.7 cm²/sec and cm²/sec, 0.9 cm²/sec and 1 cm²/sec, 1cm²/sec and 1.1 cm²/sec, 1.1 cm²/sec and 1.3 cm²/sec, 1.3 cm²/sec and1.6 cm²/sec, 1.6 cm²/sec and 2 cm²/sec, or 2 cm²/sec and 3 cm²/sec; OR

the sample to non-sample thermal mass ratio is in a range of between 0.2to 0.5, 0.5 to 0.7, 0.7 to 1, 1 to 1.5, 1.5 to 5, 5 to 10, 10 to 30, 30to 50, or 50 to 100.

NN1. The device, apparatus, system or method of any prior embodiments,wherein the device comprises a heating layer and a cooling layer, wherethe cooling layer has an area larger than that heating zone.

NN2. The device, apparatus, system or method of any prior embodiments,wherein the device comprises one heating/cooling layer, where thecooling zone has an area larger than that heating zone.

NN3. The device, apparatus, system or method of any prior embodiments,wherein the device comprises a cooling layer that has a high thermalconductivity (50 W/(m²·-K)) and an area larger than lateral area of arelevant sample.

NN4. The device, apparatus, system or method of any prior embodiments,wherein the device comprises a cooling layer that has a high thermalconductivity (greater than 50 W/(m²·K)(m·K)) and an area larger thanlateral area of a relevant sample by a factor of 2 to 40.

NN5. The device, apparatus, system or method of any prior embodiments,wherein the device comprises a cooling layer that has (i) a high thermalconductivity (greater than 50 W/(m·K)), and (ii) thermal radiationenhancement layer (specify the thermal radiation).

NN6. The device, apparatus, system or method of any prior embodiments,wherein the device comprises a cooling layer that has (i) a high thermalconductivity (greater than 50 W/(m·K)), and (ii) thermal radiationenhancement layer, and (iii) an area larger than lateral area of arelevant sample.

NN7. The device, apparatus, system or method of any prior embodiments,wherein the device comprises a cooling layer that has (i) a high thermalconductivity (greater than 50 W/(m·K)), and (ii) thermal radiationenhancement layer, and (iii) an area larger than lateral area of arelevant sample by a factor of 1.5 to 100.

NN8. The device, apparatus, system or method of any prior embodiments,wherein the device comprises a cooling zone has a thermal radiationenhancement layer that has an average light absorption coefficient of70% over the wavelength range.

NN9. The device, apparatus, system or method of any prior embodiments,wherein the device comprises a cooling zone has a thermal conductivitymultiplying its thickness in the range of 6×10⁻⁵ W/K to 3×10⁻⁴ W/K.

NN10. The device, apparatus, system or method of any prior embodiments,wherein the device comprises a cooling zone comprises a gold layer of athickness in the range of 200 nm to 800 nm.

NN11. The device, apparatus, system or method of any prior embodiments,wherein the device comprises a thermal conductivity multiplying itsthickness in the range of 6×10⁻⁵ W/K to 3×10⁻⁴ W/K.

NN12. The device, apparatus, system or method of any prior embodiments,wherein the device comprises a cooling layer that:

has a high thermal conductivity (greater than 50 W/(m·K)),

comprises thermal radiation enhancement layer that has an average lightabsorption coefficient of 70% over the wavelength range;

has an area larger than lateral area of a relevant sample by a factor of1.5 to 100; and

has a thermal conductivity multiplying its thickness in the range of6×10-5 W/K to 3×10-4 W/K.

NN13. The device, apparatus, system or method of any prior embodiments,wherein the device comprises a cooling zone (layer) has thermalconductivity times its thickness of 6×10⁻⁵ W/K, 9×10⁻⁵ W/K, 1.2×10⁻⁴W/K, 1.5×10⁻⁴ W/K, 1.8×10⁻⁴ W/K, 2.1×10⁻⁴ W/K, 2.7×10⁻⁴ W/K, 3×10⁻⁴ W/K,1.5×10⁻⁴ W/K, or in a range between any of the two values.

NN14. The device, apparatus, system or method of any prior embodiments,wherein the device comprises a cooling zone (layer) has thermalconductivity times its thickness in a range of 6×10⁻⁵ W/K to 9×10⁻⁵ W/K,9×10⁻⁵ W/K to 1.5×10⁻⁴ W/K, 1.5×10⁻⁴ W/K to 2.1×10⁻⁴ W/K, 2.1×10⁻⁴ W/Kto 2.7×10⁻⁴ W/K, 2.7×10⁻⁴ W/K to 3×10⁻⁴ W/K, or 3×10⁻⁴ W/K to 1.5×10⁻⁴W/K.

NN15. The device, apparatus, system or method of any prior embodiments,wherein the device comprises cooling zone (layer) has thermalconductivity times its thickness in a range of 9×10⁻⁵ W/K to 2.7×10⁻⁴W/K, 9×10⁻⁵ W/K to 2.4×10⁻⁴ W/K, 9×10⁻⁵ W/K to 2.1×10⁻⁴ W/K, or 9×10⁻⁵W/K to 1.8×10⁻⁴ W/K.

NN16. The device, apparatus, system or method of any prior embodiments,wherein the device comprises cooling zone comprises a gold layer of athickness in the range of 200 nm to 800 nm. In another embodiment, acooling zone comprises a gold layer of a thickness in the range of 300nm to 700 nm.

NN17. The device, apparatus, system or method of any prior embodiments,wherein in the device the materials between the heating zone and therelevant sample has a thermal conductivity and a thickness configured tohave a conductance per unit area that is equal to or larger than 1,000W/(m²·K), 2000 W/(m²·K), 3,000 W/(m²·K), 4000 W/(m²·K), 5000 W/(m²·K),7,000 W/(m²·K), 10,000 W/(m²·K), 20,000 W/(m²·K), 50,000 W/(m²·K),50,000 W/(m²·K), 100,000 W/(m²·K), or in a range of any the values.

NN18. The device, apparatus, system or method of any prior embodiments,wherein a preferred conductance per unit area of the material betweenthe heating zone and the relevant sample is in a range of 1,000 W/(m²·K)to 2,000 W/(m²·K), 2,000 W/(m²·K) to 4000 W/(m²·K), 4,000 W/(m²·K) to10,000 W/(m²·K), or 10,000 W/(m²·K) to 100,000 W/(m²·K).

NN19. The device, apparatus, system or method of any prior embodiments,wherein there is zero distance between the heating zone and the relevantsample, and hence an infinity for the conductance per unit area of thematerial between the heating zone and the relevant sample.

NN20. The device, apparatus, system or method of any prior embodiments,wherein the heating layer or the cooling layer is separated from arelevant sample by a thin plastics plate (or film) which has a thermalconductivity in the range of 0.1 to 0.3 W/(m·K), and the thin plasticlayer has a thickness of 0 nm, 10 nm, 50 nm, 100 nm, 200 nm, 300 nm, 400nm, 500 nm, 1 um, 2.5 um, 5 um, 10 um, 25 um, 50 um, 75 nm 100 um, 150um, or in a range between any of the two values

NN21. The device, apparatus, system or method of any prior embodiments,wherein the thin plastic plate (or film) that separate the relevantsample from the heating layer or the cooling layer has thickness in arange between 0 nm and 100 nm, 100 nm and 500 nm, 500 nm and 1 um, 1 umand 5 um, 5 um and 10 um, 10 um and 25 um, 25 um and 50 um, 50 um and 75um, 75 um and 100 um, or 100 um and 150 um.

NN22. The device, apparatus, system or method of any prior embodiments,wherein the thin plastic plate (or film) that separates the relevantsample from the heating layer or the cooling layer has thickness of 0.1um, 0.5 um, 1 um, 5 um, 10 um, 20 um, 25 um, or a range between any twovalues.

NN23. The device, apparatus, system or method of any prior embodiments,wherein the area of the heating zone is only a fraction of the area ofthe cooling zone or area, and the area of the cooling zone (layer) islarger than the area of the heating zone by a factor of 1.1, 1.5, 2, 3,4, 5, 10, 20, 30, 40, 50, 70, 100, 200, 300, 400, 500, 600, 700, 800,800, 1,000, 5000, 10,000, 100,000, or in a range between any of the twovalues.

NN24. The device, apparatus, system or method of any prior embodiments,wherein the cooling zone (layer) has an area that is larger than thelateral area of the hearing zone (layer) by a factor in a range of 1.1to 1.5, 1.5 to 5, 5 to 10, 10 to 50, 50 to 100, 100 to 500, 500 to1,000, 1000, to 10,000, or 10,000 to 100,000.

NN25. The device, apparatus, system or method of any prior embodiments,wherein the cooling zone (layer) has an area that is larger than thelateral area of the relevant sample by a factor of 1.5, 2, 3, 4, 5, 10,20, 50, 70, 100, 200, 300, 400, 500, 600, 700, 800, 800, 1,000, 2000,5000, 10,000, 100,000, or in a range between any of the two values.

NN26. The device, apparatus, system or method of any prior embodiments,wherein the cooling zone (layer) has an area that is larger than thelateral area of the relevant sample by a factor in a range of 1.5 to 5,5 to 10, 10 to 50, 50 to 100, 100 to 500, 500 to 1,000, 1000, to 10,000,or 10,000 to 100,000.

NN27. The device, apparatus, system or method of any prior embodiments,wherein the first plate or the second plate has a thickness of 10 nm,100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 1 um, 2.5 um, 5 um, 10 um, 25um, 50 um, 100 um, 200 um, or 500 um, 1000 um, or in a range between anyof the two values.

NN28. The device, apparatus, system or method of any prior embodiments,wherein the first plate and the second plate can have the same thicknessor a different thickness, and can be made of the same materials ordifferent materials.

NN29. The device, apparatus, system or method of any prior embodiments,wherein the first plate or the second plate has a thickness in a rangeof between 10 nm and 500 nm, 500 nm and 1 um, 1 um and 2.5 um, 2.5 umand 5 um, 5 um and 10 um, 10 um and 25 um, 25 um and 50 um, 50 um and100 um, 100 um and 200 um, or 200 um and 500 um, or 500 um and 1000 um.

NN30. The device, apparatus, system or method of any prior embodiments,wherein the first plate and second plates are plastic, a thin glass, ora material with similar physical properties. The first plate or secondplate have a thickness of 100 nm, 500 nm, 1 um, 5 um, 10 um, 25 um, 50um, 100 um, 175 um, 250 um, or in a range between any of the two values.

NN31. The device, apparatus, system or method of any prior embodiments,wherein the ratio of the average lateral size of the relevant samplevolume to the diffusion length of the reagent during the time forthermal cycling or a reaction is equal to or larger than 5, 6, 7, 10,15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, 1,000,5,000, 10,000, 100,000, or in a range between any two values.

NN32. The device, apparatus, system or method of any prior embodiments,wherein the ratio of the average lateral size of the relevant samplevolume to the diffusion length of the reagent during the time forthermal cycling or a reaction is in a range of 5 to 10, 10 to 30, 30 to60, 6 to 100, 100 to 200, 200 to 500, 500 to 1,000, 1,000 to 5000, 5,000to 10,000, or 10,000 to 100,000.

NN33. The device, apparatus, system or method of any prior embodiments,wherein the ratio of the average lateral size of the relevant samplevolume to the diffusion length of the reagent during the time forthermal cycling or a reaction is in a range of 5 to 10, 10 to 30, 30 to60, 6 to 100, 100 to 200, 200 to 500, 500 to 1,000, 1,000 to 5,000,5,000 to 10,000, or 10,000 to 100,000.

NN34. The device, apparatus, system or method of any prior embodiments,wherein the average lateral dimension of the relevant volume is 1 mm, 2mm, 3 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm 10 mm, 12 mm, 15 mm, 20 mm, 30mm, 40 mm, 50 mm, 70 mm, 100 mm, 200 mm, or in a range between any twovalues.

NN35. The device, apparatus, system or method of any prior embodiments,wherein the average lateral dimension of the relevant volume is in arange of 1 mm to 5 mm, 5 mm to 10 mm, 10 mm to 20 mm, 20 mm to 40 mm, 40mm to 70 mm, 70 mm to 100 mm, or 100 mm to 200 mm.

NN36. The device, apparatus, system or method of any prior embodiments,wherein the average lateral dimension of the relevant volume is in arange of 1 mm to 5 mm, 1 mm to 10 mm, or 5 mm to 20 mm.

NN37. The device, apparatus, system or method of any prior embodiments,wherein the thermal radiation enhancement surface has a high averagelight absorptance (e.g. the black paint used in our experiments). Incertain embodiments, the cooling zone has a surface that has an averagelight absorptance of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, or ina range between any of the two values.

NN38. The device, apparatus, system or method of any prior embodiments,wherein the cooling zone has a surface that has an average lightabsorptance in a range of 30% to 40%, 40% to 60%, 60% to 80% to 90%, or90% to 100%.

NN39. The device, apparatus, system or method of any prior embodiments,wherein the cooling zone has a surface that has an average lightabsorptance in a range of 30% to 100%, 50% to 100%, 70% to 100%, or 80%to 100%.

NN40. The device, apparatus, system or method of any prior embodiments,wherein the cooling zone has a surface that has an average lightabsorptance of a value given above by averaging over a wavelength range400 nm to 800 nm, 700 nm to 1500 nm, 900 nm to 2,000 nm, or 2,000 nm to20,000 nm.

NN41. The device, apparatus, system or method of any prior embodiments,wherein the black paints are polymer mixtures that look black by humaneyes. A black paint include, but not limited to, a mixture of polymersand nanoparticles. One example of the nanoparticles is black carbonnanoparticle, carbon, nanotubes, graphite particles, graphene, metalnanoparticles, semiconductor nanoparticles, or a combination thereof.

NN42. The device, apparatus, system or method of any prior embodiments,wherein the plasmonic structures include nanostructured plasmonicstructures.

NN43. The device, apparatus, system or method of any prior embodiments,wherein a cooling plate comprise a layer of high thermal conductivitymetal (50 W/(m·K) or higher) with a surface thermal radiationenhancement layer. In some embodiments, the surface thermal radiationenhancement layer has a low lateral thermal conductance, which is due toeither ultrathin layer, low thermal conductivity, or both.

NN44. The device, apparatus, system or method of any prior embodiments,wherein thermal radiative cooling is achieved by increasing the area ofradiative cooling layer (i.e. a high-K material, unless statedotherwise), and the radiative cooling layer area is larger than thelateral area of the relevant sample by a factor of 1.2, 1.5, 2, 3, 4, 5,10, 20, 30, 40, 50, 60, 70, 80 100, 200, 300, 400, 500, 600, 700, 800,800, 1,000, 2,000, 5,000, 10,000, 100,000, or in a range between any ofthe two values.

NN45. The device, apparatus, system or method of any prior embodiments,wherein the radiative cooling zone (layer) has an area that is largerthan the lateral area of the relevant sample by a factor in a range of1.2 to 3, 3 to 5, 5 to 10, 10 to 50, 50 to 100, 100 to 500, 500 to1,000, 1000, to 10,000, or 10,000 to 100,000.

NN46. The device, apparatus, system or method of any prior embodiments,wherein the ratio of the thermal radiation cooling by the cooling zone(layer) to the total cooling of the sample and sample holder during athermal cycling is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, orin a range between any of the two values.

NN47. The device, apparatus, system or method of any prior embodiments,wherein the ratio of the thermal radiation cooling by the cooling zone(layer) to the total cooling of the sample and sample holder during athermal cycling is in a range of between 10% and 20%, 20% and 30%, 30%and 40%, 40% and 50%, 50% and 60%, 60% and 70%, 70% and 80%, 80% and90%, or 90% and 99%.

NN48. The device, apparatus, system or method of any prior embodiments,wherein the KC ratio materials for the heating layer is equal to orhigher than 0.1 cm²/sec, 0.2 cm²/sec, 0.3 cm²/sec, 0.4 cm²/sec, 0.5cm²/sec, 0.6 cm²/sec, 0.7 cm²/sec, 0.8 cm²/sec, 0.9 cm²/sec, 1 cm²/sec,1.1 cm²/sec, 1.2 cm²/sec, 1.3 cm²/sec, 1.4 cm²/sec, 1.5 cm²/sec, 1.6cm²/sec, 2 cm²/sec, 3 cm²/sec, or in a range between any of the twovalues.

NN49. The device, apparatus, system or method of any prior embodiments,wherein the KC ratio for the heating layer is in a range of between 0.5cm²/sec and 0. cm²/sec, 0.7 cm²/sec and 0.9 cm²/sec, 0.9 cm²/sec and 1cm²/sec, 1 cm²/sec and 1.1 cm²/sec, 1.1 cm²/sec and 1.3 cm²/sec, 1.3cm²/sec and 1.6 cm²/sec, 1.6 cm²/sec and 2 cm²/sec, or 2 cm²/sec and 3cm²/sec.

NN50. The device, apparatus, system or method of any prior embodiments,wherein a thermal radiation enhancement surface(s) will be used (on oneside or both side of the heating zone). A thermal radiation absorptionenhancement surface can be achieved by directly modify the structures ofthe surface (e.g. patterning nanostructures), coating a high thermalradiation materials (e.g. coating a black paint), or both.

NN51. The device, apparatus, system or method of any prior embodiments,wherein the thermal radiation enhancement surface has a high averagelight absorptance (e.g. the black paint used in our experiments). Incertain embodiments, the heating zone has a surface that has an averagelight absorptance of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, or ina range between any of the two values.

NN52. The device, apparatus, system or method of any prior embodiments,wherein the heating zone has a surface that has an average lightabsorptance in a range of 30% to 40%, 40% to 60%, 60% to 80% to 90%, or90% to 100%.

NN53. The device, apparatus, system or method of any prior embodiments,wherein the heating zone has a surface that has an average lightabsorptance in a range of 30% to 100%, 50% to 100%, 70% to 100%, or 80%to 100%.

NN54. The device, apparatus, system or method of any prior embodiments,wherein the heating zone has a surface that has an average lightabsorptance of a value given above by averaging over a wavelength range400 nm to 800 nm, 700 nm to 1,500 nm, 900 nm to 2,000 nm, or 2,000 nm to20,000 nm.

NN55. The device, apparatus, system or method of any prior embodiments,wherein the LVS ratio for sample is 5, 10, 20, 50, 70, 100, 200, 300,400, 500, 600, 700, 800, 800, 1,000, 2,000, 5,000, 10,000, 100,000, orin a range between any of the two values.

NN56. The device, apparatus, system or method of any prior embodiments,wherein the LVS ratio for sample is in a range of 5 to 10, 10 to 50, 50to 100, 100 to 500, 500 to 1,000, 1,000, to 10,000, or 10,000 to100,000,

NN57. The device, apparatus, system or method of any prior embodiments,wherein the sample has a lateral dimension of 15 mm and a thickness of30 um, hence an LVS for the sample of 500.

NN58. The device, apparatus, system or method of any prior embodiments,wherein the thickness of the relevant sample is reduced (which also canhelp sample heating speed), and the relevant sample has a thickness of0.05 um, 0.1 um, 0.2 um, 0.5 um, 1 um, 2 um, 5 um, 10 um, 20 um, 30 um,40 um, 50 um, 60 um, 70 um, 80 um, 90 um, 100 um, 200 um, 300 um, or ina range between any of the two values.

NN59. The device, apparatus, system or method of any prior embodiments,wherein the relevant sample has a thickness in a range between 0.05 umand 0.5 um, 0.5 um and 1 um, 1 um and 5 um, 5 um and 10 um, 10 um and 30um, 30 um and 50 um, 50 um and 70 um, 70 um and 100 um, 100 um and 200um, or 200 um and 300 um.

NN60. The device, apparatus, system or method of any prior embodiments,wherein the KC ratio materials for the cooling layer is equal to orhigher than 0.1 cm²/sec, 0.2 cm²/sec, 0.3 cm²/sec, 0.4 cm²/sec, 0.5cm²/sec, 0.6 cm²/sec, 0.7 cm²/sec, 0.8 cm²/sec, 0.9 cm²/sec, 1 cm²/sec,1.1 cm²/sec, 1.2 cm²/sec, 1.3 cm²/sec, 1.4 cm²/sec, 1.5 cm²/sec, 1.6cm²/sec, 2 cm²/sec, 3 cm²/sec, or in a range between any of the twovalues.

NN61. The device, apparatus, system or method of any prior embodiments,wherein the KC ratio for the cooling layer is in a range of between 0.5cm²/sec and 0.7 cm²/sec, 0.7 cm²/sec and 0.9 cm²/sec, 0.9 cm²/sec and 1cm²/sec, 1 cm²/sec and 1.1 cm²/sec, 1.1 cm²/sec and 1.3 cm²/sec, 1.3cm²/sec and 1.6 cm²/sec.

NN62. The device, apparatus, system or method of any prior embodiments,wherein a high thermal conductivity (i.e. high-K) material is used forthe cooling layer, and the high-K material has a thermal conductivity ofequal to or larger than 50 W/(m·K), 80 W/(m·K), 100 W/(m·K), 150W/(m·K), 200 W/(m·K), 250 W/(m·K), 300 W/(m·K), 350 W/(m·K), 400W/(m·K), 450 W/(m·K), 500 W/(m·K), or in a range between any of the twovalues.

NN63. The device, apparatus, system or method of any prior embodiments,wherein the sample to non-sample thermal mass ratio (NSTM ratio) is 0.1,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 1, 1.5, 2, 3, 4, 5, 10, 20, 30, 40, 50,60, 70, 100, 200, 300, 1,000, 4,000, or in a range between any of thetwo values.

NN64. The device, apparatus, system or method of any prior embodiments,wherein the sample to non-sample thermal mass ratio (NSTM ratio) is in arange of between 0.1 to 0.2, 0.2 to 0.5, 0.5 to 0.7, 0.7 to 1, 1 to 1.5,1.5 to 5, 5 to 10, 10 to 30, 30 to 50, 50 to 100, 100 to 300, 300 to1,000, or 1,000 to 4,000.

NN65. The device, apparatus, system or method of any prior embodiments,wherein the device is configured to make the sample to non-samplethermal mass ratio high, one need to keep the area thermal mass of thenon-sample low, which in turn, needs to make the plates and theheating/cooling layer thin, and/or the volume specific heat low.

NN66. The device, apparatus, system or method of any prior embodiments,wherein the device comprises a thin material that has multi-layers ormixed materials. For examples, a carbon fiber layer(s) with plasticsheets or carbon mixed with plastics, which can have a thickness of 0.1um, 0.2 um, 0.5 um, 1 um, 2 um, 5 um, 10 um, 25 um, 50 um, or in a rangebetween any of the two values.

NN67. The device, apparatus, system or method of any prior embodiments,wherein the relevant volume of the sample is 0.001 ul, 0.005 ul, 0.01ul, 0.02 ul, 0.05 ul, 0.1 ul, 0.2 ul, 0.5 ul, 1 ul, 2 ul, 5 ul, 10 ul,20 ul, 30 uL, 50 ul, 100 ul, 200 ul, 500 ul, 1 ml, 2 ml, 5 ml, or in arange between any of the two values.

NN68. The device, apparatus, system or method of any prior embodiments,wherein the relevant sample volume is in a range of 0.001 uL to 0.1 uL,0.1 um to 2 uL, 2 uL to 10 uL, 10 uL to 30 uL, 30 uL to 100 uL, 100 uLto 200 uL, or 200 uL to 1 mL.

NN69. The device, apparatus, system or method of any prior embodiments,wherein the relevant sample volume is in a range of 0.001 uL to 0.1 uL,0.1 um to 1 uL, 0.1 uL to 5 uL, or 0.1 uL to 10 uL.

NN70. The device, apparatus, system or method of any prior embodiments,wherein the ratio of the relevant sample to entire sample volume (REratio) is 0.01%, 0.05%, 0.1%, 0.5%, 0.1%, 0.5%, 1%, 5%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 100%, or in a range between any of the twovalues.

NN71. The device, apparatus, system or method of any prior embodiments,wherein the RE ratio is in a range of between 0.01% and 0.1%, 0.1% and1%, 1% and 10%, 10% and 30%, 30% and 60%, 60% and 90%, or 90% and 100%.

NN72. The device, apparatus, system or method of any prior embodiments,wherein the area of the heating zone is only a fraction of the samplelateral area, and the fraction (i.e. the ratio of the heating zone tothe sample lateral area) is 0.01%, 0.05%, 0.1%, 0.5%, 0.1%, 0.5%, 1%,5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or in a rangebetween any of the two values.

NN73. The device, apparatus, system or method of any prior embodiments,wherein the ratio of the heating zone area to the sample lateral area isin a range of between 0.01% and 0.1%, 0.1% and 1%, 1% and 10%, 10% and30%, 30% and 60%, 60% and 90%, or 90% and 99%.

NN74. The device, apparatus, system or method of any prior embodiments,wherein the scaled thermal conduction ratio (STM ratio) is 2 or larger,5 or larger, 10 or larger, 20 or larger, 30 or larger, 40 or larger, 50or larger, 100 or larger, 1,000 or larger, 10,000 or larger, 10,000 orlarger, or in a range between any of the two values.

NN75.1 The device, apparatus, system or method of any prior embodiments,wherein the scaled thermal conduction ratio (STM ratio) is in a range ofbetween 10 to 20, 30 to 50, 50 to 70, 70 to 100, 100 to 1,000, 1,000 to10,000, or 10,000 to 1,000,000.

NN75.2 The device, apparatus, system or method of any prior embodiments,wherein the scaled thermal conduction ratio (STM ratio) is in a range ofbetween 10 to 20, 30 to 50, 50 to 70, 70 to 100, 100 to 1,000, 1,000 to10,000, or 10,000 to 1,000,000; and the cooling zone (layer) has thermalconductivity times its thickness of 6×10⁻⁵ W/K, 9×10⁻⁵ W/K, 1.2×10⁻⁴W/K, 1.5×10⁻⁴ W/K, 1.8×10⁻⁴ W/K, 2.1×10⁻⁴ W/K, 2.7×10⁻⁴ W/K, 3×10⁻⁴ W/K,1.5×10⁻⁴ W/K, or in a range between any of the two values.

NN75.3 The device, apparatus, system or method of any prior embodiments,wherein the scaled thermal conduction ratio (STM ratio) is in a range ofbetween 20 to 80.

NN76. The device, apparatus, system or method of any prior embodiments,wherein

the lateral to vertical size (LVS) ratio for relevant sample is 5, 10,20, 50, 70, 100, 200, 300, 400, 500, 600, 700, 800, 800, 1,000, 2000,5000, 10,000, 100,000, or in a range between any of the two values.

NN77. The device, apparatus, system or method of any prior embodiments,wherein

the LVS ratio for relevant sample is in a range of 5 to 10, 10 to 50, 50to 100, 100 to 500, 500 to 1,000, 1,000, to 10,000, or 10,000 to100,000.

NN78. The device, apparatus, system or method of any prior embodiments,wherein the thickness of the relevant sample is reduced (which also canhelp sample heating speed), and the relevant sample has a thickness of0.05 um, 0.1 um, 0.2 um, 0.5 μm, 1 um, 2 um, 5 um, 10 um, 20 um, 30 um,40 um, 50 um, 60 um, 70 um, 80 um, 90 um, 100 um, 200 um, 300 um, or ina range between any of the two values.

NN78. The device, apparatus, system or method of any prior embodiments,wherein the relevant sample has a thickness in a range between 0.05 umand 0.5 μm, 0.5 um and 1 um, 1 um and 5 um, 5 um and 10 um, 10 um and 30um, 30 um and 50 um, 50 um and 70 um, 70 um and 100 um, 100 um and 200um, or 200 um and 300 um.

OO1. A device, comprising:

a first plate comprising a polymer material and having a thickness lessthan or equal to 100 μm;

a second plate comprising a polymer material and having a thickness lessthan or equal to 100 μm; and

a heating/cooling layer disposed on either the first plate or the secondplate, the heating/cooling layer having a thermal conductivity between6×10⁻⁵ W/K multiplied by the thickness of the heating/cooling layer and1.5×10⁻⁴ W/K multiplied by the thickness of the heating/cooling layer,

wherein the first plate and the second plate face each other in aparallel arrangement, and are separated from each other by a distance,and wherein the first plate and the second plate are configured toreceive a fluid sample sandwiched between the first plate and the secondplate.

OO2. A device, comprising:

a first plate;

a second plate having a thickness less than or equal to 100 μm, whereinthe second plate is separated from the first plate in a parallelarrangement by a distance less than or equal to the thickness of thesecond plate; and

a heating/cooling layer disposed on either the first plate or the secondplate,

wherein the heating/cooling layer is configured to receiveelectromagnetic radiation such that at least a portion of a liquidsample sandwiched between the first plate and the second plate is heatedat a rate of at least 30° C./sec.

OO3. A device, comprising:

a first plate;

a second plate having a thickness less than or equal to 100 μm, whereinthe second plate is separated from the first plate in a parallelarrangement by a distance less than or equal to the thickness of thesecond plate; and

a heating/cooling layer disposed on either the first plate or the secondplate,

wherein at least a portion of a liquid sample sandwiched between thefirst plate and the second plate is cooled at a rate of at least 30°C./sec when the heating/cooling layer is not receiving electromagneticradiation generated by an optical source.

OO4. A device, comprising:

a first plate;

a second plate having a thickness less than or equal to 100 μm, whereinan inner surface of the second plate is separated from an inner surfaceof the first plate in a parallel arrangement by a distance less than orequal to the thickness of the second plate;

a heating/cooling layer disposed on the inner surface or on an outersurface of the second plate; and

a layer of reagents dried on the inner surface of the first plate.

OO5. The device of any of OO1-OO4 embodiments, further comprising alight absorbing layer disposed on the heating/cooling layer, wherein thelight absorbing layer has an average light absorptance of at least 30%.

OO6. The device of OO5, wherein the light absorbing layer comprisesblack paint.

OO7. The device of any of OO1-OO6 embodiments, wherein the first plateis movable relative to the second plate.

OO8. The device of any of OO1-OO7 embodiments, wherein a thickness ofthe heating/cooling layer is less than or equal to 3 μm.

OO9. The device of any of OO1-OO8 embodiments, wherein at least one ofthe first plate and the second plate has an area across its majorsurface of about 400 mm².

OO10. The device of any of OO1-OO9 embodiments, further comprising aplurality of spherical spacers disposed between the first plate and thesecond plate.

OO11. The device of any of OO1-OO9 embodiments, further comprising aplurality of spacers having a height of about 10 um, wherein theplurality of spacers are disposed between the first plate and the secondplate.

OO12. The device of any of OO1-OO11 embodiments, wherein the distancebetween the first plate and the second plate is less than or equal to100 μm.

OO13. The device of any of OO1-OO12 embodiments, further comprising ahinge configured to connect the first plate with the second plate, andcoupled to an edge of the first plate or the second plate.

OO14. The device of any of OO1-OO13 embodiments, wherein the at least aportion of the liquid sample comprises a volume of the sample along apath of the electromagnetic radiation.

OO15. The device of any of OO1-OO14 embodiments, wherein the at least aportion of the liquid sample comprises a volume of the sample that isadjacent to the heating/cooling layer.

OO16. The device of OO4, wherein the layer of dried reagents comprisesreagents used for nucleic acid amplification.

PP1. A system, comprising:

a device, comprising:

a first plate comprising a polymer material and having a thickness lessthan or equal to 100 μm,

a second plate comprising a polymer material and having a thickness lessthan or equal to 100 μm, wherein the second plate is separated from thefirst plate in a parallel arrangement by a distance less than or equalto the thickness of the second plate,

a heating/cooling layer disposed on either the first plate or the secondplate, the heating/cooling layer having a thickness and a thermalconductivity between 6×10⁻⁵ W/K multiplied by the thickness of theheating/cooling layer and 1.5×10⁻⁴ W/K multiplied by the thickness ofthe heating/cooling layer, and

a support frame configured to support at least one of the first plateand the second plate;

a housing having a first opening configured to receive the device and atleast one other opening;

an optical source configured to direct electromagnetic radiation towardsthe heating/cooling layer,

wherein the heating/cooling layer is configured to absorb at least aportion of the electromagnetic radiation such that at least a portion ofa liquid sample sandwiched between the first plate and the second plateis heated at a rate of at least 30° C./sec, and

wherein at least the portion of the liquid sample sandwiched between thefirst plate and the second plate is cooled at a rate of at least 30°C./sec when the heating/cooling layer is not receiving theelectromagnetic radiation generated by the optical source, and whereinthe system consumes less than 500 mW of power.

PP2. A system, comprising:

a device, comprising:

a first plate,

a second plate having a thickness less than or equal to 100 μm, whereinthe second plate is separated from the first plate in a parallelarrangement by a distance less than or equal to the thickness of thesecond plate,

a heating/cooling layer disposed on either the first plate or the secondplate, and

a support frame configured to support at least one of the first plateand the second plate; and

an optical source configured to direct electromagnetic radiation towardsthe heating/cooling layer,

wherein at least a portion of a liquid sample sandwiched between thefirst plate and the second plate is cooled at a rate of at least 30°C./sec when the heating/cooling layer is not receiving theelectromagnetic radiation generated by the optical source.

PP3. A system, comprising:

a device, comprising:

a first plate,

a second plate having a thickness less than or equal to 100 μm, whereinthe second plate is separated from the first plate in a parallelarrangement by a distance less than or equal to the thickness of thesecond plate,

a heating/cooling layer disposed on either the first plate or the secondplate; and

an optical source configured to direct electromagnetic radiation towardsthe heating/cooling layer, wherein the system consumes less than 500 mWof power.

PP4. A system, comprising:

a device, comprising:

a first plate,

a second plate having a thickness less than or equal to 100 μm, whereinthe second plate is separated from the first plate in a parallelarrangement by a distance less than or equal to the thickness of thesecond plate,

a heating/cooling layer disposed on either the first plate or the secondplate, and

a support frame configured to support at least one of the first plateand the second plate;

a housing having a first opening configured to receive the device and atleast one other opening; and

an optical source configured to direct electromagnetic radiation throughthe at least one other opening of the housing and towards theheating/cooling layer,

wherein a liquid sample sandwiched between the first plate and thesecond plate is cooled at a rate of at least 30° C./sec when theheating/cooling layer is not receiving the electromagnetic radiationgenerated by the optical source.

PP5. The system of any one of PP1-PP4 embodiments, wherein the devicefurther comprises a light absorbing layer disposed on theheating/cooling layer, wherein the light absorbing layer has an averagelight absorptance of at least 30%.

PP6. The system of PP5, wherein the light absorbing layer comprisesblack paint.

PP7. The system of any one of PP1-PP6 embodiments, wherein the firstplate is movable relative to the second plate.

PP8. The system of any one of PP1-PP7 embodiments, wherein a thicknessof the heating/cooling layer is less than or equal to 3 μm.

PP9. The system of any one of PP1-PP8 embodiments, wherein at least oneof the first plate and the second plate has an area across its majorsurface of about 400 mm².

PP10. The system of any one of PP1-PP9 embodiments, wherein the opticalsource comprises a light emitting diode (LED.)

PP11. The system of PP10, wherein the LED comprises a blue LED.

PP12. The system of any one of PP1-PP11 embodiments, further comprisingan optical pipe configured to guide the electromagnetic radiation fromthe optical source to the heating/cooling layer.

PP13. The system of PP1 or PP4, wherein the at least one other openingof the housing is configured to be aligned over at least the portion ofthe liquid sample sandwiched between the first plate and the secondplate when the device is placed within the housing via the firstopening.

PP14. The system of any one of PP1-PP13 embodiments, wherein the supportframe is configured to support at least the first plate or the secondplate along a perimeter of the first plate or second plate.

QQ1. A method of using a device, comprising:

placing a second plate over a first plate such that a fluidic sample issandwiched between the first plate and the second plate at a thicknessdetermined by one or more spacers located on at least one of the firstplate and the second plate;

activating a heat source configured to radiate electromagnetic radiationtowards a heating layer located on either the first plate or the secondplate; and

heating, using at least the heating layer, at least a portion of thefluidic sample at a rate of at least 30° C./sec.

QQ2. A method of using a device, comprising:

placing a second plate over the first plate such that a fluidic sampleis sandwiched between the first plate and the second plate at athickness determined by one or more spacers located on at least one ofthe first plate and the second plate;

activating, for a given time period, a heat source configured to radiateelectromagnetic radiation towards a heating/cooling layer located oneither the first plate or the second plate;

deactivating the heat source after the given time period, wherein atleast a portion of the fluidic sample cools at a rate of at least 30°C./sec after the deactivating.

QQ3. A method of using a device, comprising:

placing a second plate over the first plate such that a fluidic sampleis sandwiched between the first plate and the second plate at athickness determined by one or more spacers located on at least one ofthe first plate and the second plate;

activating a heat source configured to radiate electromagnetic radiationtowards a heating layer located on either the first plate or the secondplate, wherein the heat source consumes less than 500 mW of power; and

heating, using at least the heating layer, at least a portion of thefluidic sample.

QQ4. The method of any one of QQ1-QQ3 embodiments, wherein the firstplate or the second plate further comprises a light absorbing layerdisposed on the heating layer, wherein the light absorbing layer has anaverage light absorptance of at least 30%.

QQ5. The method of QQ4, wherein the light absorbing layer comprisesblack paint.

QQ6. The method of any one of QQ1-QQ5 embodiments, further comprisingclosing the second plate over the first plate using a hinge connectedbetween the first plate and the second plate.

QQ7. The method of any one of QQ1-QQ6 embodiments, wherein a thicknessof the heating layer is less than or equal to 3 μm.

QQ8. The method of any one of QQ1-QQ7 embodiments, wherein at least oneof the first plate and the second plate has an area across its majorsurface of about 400 mm².

QQ9. The method of any one of QQ1-QQ8 embodiments, wherein activating aheat source comprises activating an LED to radiate light towards theheating layer.

QQ10. The method of QQ9, further comprising controlling an output of theLED based on a measured or estimated temperature of the portion of thefluidic sample.

QQ11. The method of any one of QQ1-QQ10 embodiments, further comprisingexpanding the electromagnetic radiation using a beam expander before theelectromagnetic radiation reaches the heating layer.

QQ12. The method of any one of QQ1-QQ11 embodiments, further comprisingsupporting a perimeter of either the first plate or the second plate ona support frame.

RR1. A method of amplifying nucleic acids, comprising:

depositing a fluidic sample containing nucleic acids on a first plate ofa fluidic device;

placing a second plate over the first plate such that the fluidic sampleis sandwiched between the first plate and the second plate, whereinreagents for nucleic acid amplification are present on the inner surfaceof the second plate;

activating a heat source configured to radiate electromagnetic radiationtowards a heating layer located on either the first plate or the secondplate;

heating, using at least the heating layer, at least a portion of thefluidic sample at a rate of at least 30° C./sec; and

accumulating nucleic acid amplification products in at least the portionof the fluidic sample sandwiched between the first plate and the secondplate.

RR2. A method of amplifying nucleic acids, comprising:

depositing a fluidic sample containing nucleic acids on a first plate ofa fluidic device;

placing a second plate over the first plate such that the fluidic sampleis sandwiched between the first plate and the second plate, whereinreagents for nucleic acid amplification are present on the inner surfaceof the second plate;

amplifying nucleic acids in the sample by conducting one or more PCRcycles, wherein each PCR cycle comprises a denaturing step, an annealingstep, and an elongation step;

wherein one or more of the denaturing step, the annealing step, and/orthe elongation step comprises:

activating a heat source configured to radiate electromagnetic radiationtowards a heating layer located on either the first plate or the secondplate; and

heating, using at least the heating layer, at least a portion of thefluidic sample at a rate of at least 30° C./sec.

RR3. A method of amplifying nucleic acids, comprising:

depositing a fluidic sample containing nucleic acids on a first plate ofa fluidic device;

placing a second plate over the first plate such that the fluidic sampleis sandwiched between the first plate and the second plate, whereinreagents for nucleic acid amplification are present on the inner surfaceof the second plate;

activating, for a given time period, a heat source configured to radiateelectromagnetic radiation towards a heating/cooling layer located oneither the first plate or the second plate;

deactivating the heat source after the given time period, wherein atleast a portion of the fluidic sample adjacent to the heating/coolinglayer cools at a rate of at least 30° C./sec after the deactivating; and

accumulating nucleic acid amplification products in at least the portionof the fluidic sample sandwiched between the first plate and the secondplate.

RR4. A method of amplifying nucleic acids, comprising:

depositing a fluidic sample containing nucleic acids on a first plate ofa fluidic device;

placing a second plate over the first plate such that the fluidic sampleis sandwiched between the first plate and the second plate, whereinreagents for nucleic acid amplification are present on the inner surfaceof the second plate;

amplifying nucleic acids in the sample by conducting one or more PCRcycles, wherein each PCR cycle comprises a denaturing step, an annealingstep, and an elongation step;

wherein one or more of the denaturing steps, the annealing step, and/orthe elongation step comprises:

activating a heat source configured to radiate electromagnetic radiationtowards a heating layer located on either the first plate or the secondplate; and

deactivating the heat source after the given time period, wherein atleast a portion of the fluidic sample adjacent to the heating/coolinglayer cools at a rate of at least 30° C./sec after the deactivating; and

accumulating nucleic acid amplification products in at least the portionof the fluidic sample sandwiched between the first plate and the secondplate.

RR5. A method of amplifying nucleic acids, comprising:

depositing a fluidic sample containing nucleic acids on a first plate ofa fluidic device;

placing a second plate over the first plate such that the fluidic sampleis sandwiched between the first plate and the second plate at athickness determined by one or more spacers located on at least one ofthe first plate and the second plate, wherein reagents for nucleic acidamplification are present on the inner surface of the second plate;

activating a heat source configured to radiate electromagnetic radiationtowards a heating layer located on either the first plate or the secondplate, wherein the heat source consumes less than 500 mW of power;

heating, using at least the heating layer, at least a portion of thefluidic sample; and

accumulating nucleic acid amplification products in at least the portionof the fluidic sample sandwiched between the first plate and the secondplate.

RR6. A method of amplifying nucleic acids, comprising:

depositing a fluidic sample containing nucleic acids on a first plate ofa fluidic device;

placing a second plate over the first plate such that the fluidic sampleis sandwiched between the first plate and the second plate at athickness determined by one or more spacers located on at least one ofthe first plate and the second plate, wherein reagents for nucleic acidamplification are present on the inner surface of the second plate;

amplifying nucleic acids in the sample by conducting one or more PCRcycles, wherein each PCR cycle comprises a denaturing step, an annealingstep, and an elongation step;

wherein one or more of the denaturing step, the annealing step, and/orthe elongation step comprises:

activating a heat source configured to radiate electromagnetic radiationtowards a heating layer located on either the first plate or the secondplate, wherein the heat source consumes less than 500 mW of power;

heating, using at least the heating layer, at least a portion of thefluidic sample; and

accumulating nucleic acid amplification products in at least the portionof the fluidic sample sandwiched between the first plate and the secondplate.

RR7. The method of any one of RR1-RR6 embodiments, wherein the firstplate or the second plate further comprises a light absorbing layerdisposed on the heating/cooling layer, wherein the light absorbing layerhas an average light absorptance of at least 30%.

RR8. The method of RR7, wherein the light absorbing layer comprisesblack paint.

RR9. The method of any one of RR1-RR8 embodiments, further comprisingclosing the second plate over the first plate using a hinge connectedbetween the first plate and the second plate.

RR10. The method of any one of RR1-RR9 embodiments, wherein a thicknessof the heating/cooling layer is less than or equal to 3 μm.

RR11. The method of any one of RR1-RR10 embodiments, wherein at leastone of the first plate and the second plate has an area across its majorsurface of about 400 mm².

RR12. The method of any one of RR1-RR11 embodiments, wherein activatinga heat source comprises activating an LED to radiate light towards theheating/cooling layer.

RR13. The method of RR12, further comprising controlling an output ofthe LED based on a measured or estimated temperature of the portion ofthe fluidic sample.

RR14. The method of any one of RR1-RR13 embodiments, further comprisingexpanding the electromagnetic radiation using a beam expander before theelectromagnetic radiation reaches the heating layer.

RR15. The method of any one of RR1-RR14 embodiments, further comprisingsupporting a perimeter of either the first plate or the second plate ona support frame.

SS1. A method for detecting whether a target nucleic acid sequence ispresent or absent in a sample, comprising:

depositing a fluidic sample containing nucleic acids on a first plate ofa fluidic device;

placing a second plate over the first plate such that the fluidic sampleis sandwiched between the first plate and the second plate, whereinreagents for nucleic acid amplification are present on the inner surfaceof the second plate, and wherein the reagents comprise primers that canhybridize with the target nucleic acid;

activating a heat source configured to radiate electromagnetic radiationtowards a heating layer located on either the first plate or the secondplate;

heating, using at least the heating layer, at least a portion of thefluidic sample at a rate of at least 30° C./sec; and

detecting whether the fluidic sample contains amplified product of thetarget nucleic acid sequence.

SS2. A method for detecting whether a target nucleic acid sequence ispresent or absent in a sample, comprising:

depositing a fluidic sample containing nucleic acids on a first plate ofa fluidic device;

placing a second plate over the first plate such that the fluidic sampleis sandwiched between the first plate and the second plate, whereinreagents for nucleic acid amplification are present on the inner surfaceof the second plate, and wherein the reagents comprise primers that canhybridize with the target nucleic acid;

activating, for a given time period, a heat source configured to radiateelectromagnetic radiation towards a heating/cooling layer located oneither the first plate or the second plate;

deactivating the heat source after the given time period, wherein atleast a portion of the fluidic sample adjacent to the heating/coolinglayer cools at a rate of at least 30° C./sec after the deactivating; and

detecting whether the fluidic sample contains amplified product of thetarget nucleic acid sequence.

SS3. A method for detecting whether a target nucleic acid sequence ispresent or absent in a sample, comprising:

depositing a fluidic sample containing nucleic acids on a first plate ofa fluidic device;

placing a second plate over the first plate such that the fluidic sampleis sandwiched between the first plate and the second plate, whereinreagents for nucleic acid amplification are present on the inner surfaceof the second plate, and wherein the reagents comprise primers that canhybridize with the target nucleic acid;

activating a heat source configured to radiate electromagnetic radiationtowards a heating layer located on either the first plate or the secondplate, wherein the heat source consumes less than 500 mW of power;

heating, using at least the heating layer, at least a portion of thefluidic sample; and

detecting whether the fluidic sample contains amplified product of thetarget nucleic acid sequence.

SS4. The method of any one of SS1-SS3 embodiments, wherein the firstplate or the second plate further comprises a light absorbing layerdisposed on the heating/cooling layer, wherein the light absorbing layerhas an average light absorptance of at least 30%.

SS5. The method of SS4, wherein the light absorbing layer comprisesblack paint.

SS6. The method of any one of SS1-SS5 embodiments, further comprisingclosing the second plate over the first plate using a hinge connectedbetween the first plate and the second plate.

SS7. The method of any one of SS1-SS6 embodiments, wherein a thicknessof the heating/cooling layer is less than or equal to 3 μm.

SS8. The method of any one of SS1-SS7 embodiments, wherein at least oneof the first plate and the second plate has an area across its majorsurface of about 400 mm².

SS9. The method of any one of SS1-SS8 embodiments, wherein activating aheat source comprises activating an LED to radiate light towards theheating/cooling layer.

SS10. The method of SS9, further comprising controlling an output of theLED based on a measured or estimated temperature of the portion of thefluidic sample.

SS11. The method of any one of SS1-SS10 embodiments, further comprisingexpanding the electromagnetic radiation using a beam expander before theelectromagnetic radiation reaches the heating layer.

SS12. The method of any one of SS1-SS11 embodiments, further comprisingsupporting a perimeter of either the first plate or the second plate ona support frame.

TT1. A method for detecting the presence or absence of an analyte in asample, comprising:

depositing a fluidic sample on a first plate of a fluidic device;

placing a second plate over the first plate such that the fluidic sampleis sandwiched between the first plate and the second plate, whereinreagents for detection of the analyte are present on the inner surfaceof the second plate;

activating a heat source configured to radiate electromagnetic radiationtowards a heating layer located on either the first plate or the secondplate;

heating, using at least the heating layer, at least a portion of thefluidic sample at a rate of at least 30° C./sec; and

detecting whether the fluidic sample contains the analyte.

TT2. A method for detecting the presence or absence of an analyte in asample, comprising:

depositing a fluidic sample containing on a first plate of a fluidicdevice;

placing a second plate over the first plate such that the fluidic sampleis sandwiched between the first plate and the second plate, whereinreagents for detection of the analyte are present on the inner surfaceof the second plate;

activating, for a given time period, a heat source configured to radiateelectromagnetic radiation towards a heating/cooling layer located oneither the first plate or the second plate;

deactivating the heat source after the given time period, wherein atleast a portion of the fluidic sample adjacent to the heating/coolinglayer cools at a rate of at least 30° C./sec after the deactivating; and

detecting whether the fluidic sample contains the analyte.

TT3. A method for detecting the presence or absence of an analyte in asample, comprising:

depositing a fluidic sample on a first plate of a fluidic device;

placing a second plate over the first plate such that the fluidic sampleis sandwiched between the first plate and the second plate, whereinreagents for detection of the analyte are present on the inner surfaceof the second plate;

activating a heat source configured to radiate electromagnetic radiationtowards a heating layer located on either the first plate or the secondplate, wherein the heat source consumes less than 500 mW of power;

heating, using at least the heating layer, at least a portion of thefluidic sample; and

detecting whether the fluidic sample contains the analyte.

UU1. A method for diagnosing a condition in a subject, comprising:

depositing a fluidic sample from the subject on a first plate of afluidic device;

placing a second plate over the first plate such that the fluidic sampleis sandwiched between the first plate and the second plate, whereinreagents for detection of an analyte are present on the inner surface ofthe second plate;

activating a heat source configured to radiate electromagnetic radiationtowards a heating layer located on either the first plate or the secondplate;

heating, using at least the heating layer, at least a portion of thefluidic sample at a rate of at least 30° C./sec; and

detecting whether the fluidic sample contains the analyte;

wherein presence or absence of the analyte indicates that the subjecthas the condition.

UU2. A method for diagnosing a condition in a subject, comprising:

depositing a fluidic sample from the subject on a first plate of afluidic device;

placing a second plate over the first plate such that the fluidic sampleis sandwiched between the first plate and the second plate, whereinreagents for detection of the analyte are present on the inner surfaceof the second plate;

activating, for a given time period, a heat source configured to radiateelectromagnetic radiation towards a heating/cooling layer located oneither the first plate or the second plate;

deactivating the heat source after the given time period, wherein atleast a portion of the fluidic sample adjacent to the heating/coolinglayer cools at a rate of at least 30° C./sec after the deactivating; and

detecting whether the fluidic sample contains the analyte;

wherein presence or absence of the analyte indicates that the subjecthas the condition.

UU3. A method for diagnosing a condition in a subject, comprising:

depositing a fluidic sample from the subject on a first plate of afluidic device;

placing a second plate over the first plate such that the fluidic sampleis sandwiched between the first plate and the second plate, whereinreagents for detection of an analyte are present on the inner surface ofthe second plate;

activating a heat source configured to radiate electromagnetic radiationtowards a heating layer located on either the first plate or the secondplate, wherein the heat source consumes less than 500 mW of power;

heating, using at least the heating layer, at least a portion of thefluidic sample; and

detecting whether the fluidic sample contains the analyte;

wherein presence or absence of the analyte indicates that the subjecthas the condition.

UU4. A method for diagnosing a condition in a subject, comprising:

depositing a fluidic sample from the subject on a first plate of afluidic device;

placing a second plate over the first plate such that the fluidic sampleis sandwiched between the first plate and the second plate, whereinreagents for detection of an analyte are present on the inner surface ofthe second plate;

activating a heat source configured to radiate electromagnetic radiationtowards a heating layer located on either the first plate or the secondplate;

heating, using at least the heating layer, at least a portion of thefluidic sample at a rate of at least 30° C./sec;

quantifying the amount of the analyte in the fluidic sample; and

comparing the amount to a control or reference amount of the analyte;

wherein a greater or reduced amount of the analyte in the samplecompared to the control or reference amount indicates that the subjecthas the condition.

UU5. A method for diagnosing a condition in a subject, comprising:

depositing a fluidic sample from the subject on a first plate of afluidic device;

placing a second plate over the first plate such that the fluidic sampleis sandwiched between the first plate and the second plate, whereinreagents for detection of an analyte are present on the inner surface ofthe second plate;

activating, for a given time period, a heat source configured to radiateelectromagnetic radiation towards a heating/cooling layer located oneither the first plate or the second plate;

deactivating the heat source after the given time period, wherein atleast a portion of the fluidic sample adjacent to the heating/coolinglayer cools at a rate of at least 30° C./sec after the deactivating;

quantifying the amount of the analyte in the fluidic sample; and

comparing the amount to a control or reference amount of the analyte;

wherein a greater or reduced amount of the analyte in the samplecompared to the control or reference amount indicates that the subjecthas the condition.

UU6. A method for diagnosing a condition in a subject, comprising:

depositing a fluidic sample from the subject on a first plate of afluidic device;

placing a second plate over the first plate such that the fluidic sampleis sandwiched between the first plate and the second plate, whereinreagents for detection of an analyte are present on the inner surface ofthe second plate;

activating a heat source configured to radiate electromagnetic radiationtowards a heating layer located on either the first plate or the secondplate, wherein the heat source consumes less than 500 mW of power;

heating, using at least the heating layer, at least a portion of thefluidic sample; and

quantifying the amount of the analyte in the fluidic sample; and

comparing the amount to a control or reference amount of the analyte;

wherein a greater or reduced amount of the analyte in the samplecompared to the control or reference amount indicates that the subjecthas the condition.

VV1. A kit, comprising:

a device of any one of OO embodiments; and

a pre-mixed polymerase chain reaction medium.

VV2. The kit of VV1, wherein the pre-mixed polymerase chain reactionmedium comprises: a DNA template, two primers, a DNA polymerase,deoxynucleoside triphosphates (dNTPs), a bivalent cation, a monovalentcation, and a buffer solution.

PCR and Molecule Amplification

In some embodiments, the device, apparatus, system, and/or method hereindescribed can be used for rapid molecule (e.g. nucleic acid)amplification. In certain embodiments, the device, apparatus, system,and method can be used for isothermal nucleic acid amplification. Incertain embodiments, the device, apparatus, and system can be used fornon-isothermal nucleic acid amplification.

Non-isothermal nucleic acid amplification generally requires the cycledaddition and removal of thermal energy. Many non-isothermal strategiesthat can be used for nucleic acid amplification involve the heating andcooling, to precise temperatures at precise times, of a reaction mixturethat includes one or several nucleic acids of interest (that can orcannot be chemically modified with additional agents) and reagentsnecessary to complete an amplification reaction. Non-limiting examplesof such nucleic acid amplification reactions include PCR; variants ofPCR (e.g., reverse transcriptase PCR (RT-PCR), quantitative PCR (Q-PCR),or realtime quantitative PCR (RTQ-PCR)); ligase-chain reaction (LCR);variants of LCR (e.g., reverse transcriptase LCR (RTLCR), quantitativeLCR (Q-LCR), real-time quantitative LCR (RTQ-LCR)); and digital nucleicamplification reactions (e.g., digital PCR (dPCR), digital RT-PCR(dRT-PCR), digital Q-PCR (dQ-PCR), digital RTQ-PCR (dRTQ-PCR), digitalLCR (dLCR), digital RT-LCR (dRT-LCR), digital Q-LCR (dQ-LCR), digitalRTQ-LCR (dRTQ-LCR). These nucleic acid amplification reactions, andothers, are described in more detail below.

PCR Reactions

The device, apparatus, system, and method of the current disclosure caninclude the completion of a PCR amplification reaction, or any stepcomprising a PCR amplification (e.g., denaturation, annealing,elongation, etc). In some embodiments, a sample can comprise reagentsnecessary to complete a PCR reaction. Nonlimiting examples of reagentsfor a PCR reaction include a template nucleic acid (e.g., DNA) moleculeto be amplified, a set of two primers that can hybridize with a targetsequence on the template nucleic acid, a polymerase (e.g., DNApolymerase), deoxynucleotide triphosphates (dNTPs), a buffer at a pH andconcentration suitable for a desired PCR reaction, a monovalent cation,and a divalent cation. Generally, the ratio of each reagent in thesample can vary and depend upon, for example, the amount of nucleic acidto be amplified and/or the desired amount of amplification products.Methods to determine the ratio of each reagent necessary for a PCRamplification reaction are found in, for example, U.S. Pat. Nos.4,683,202 and 4,683,195, which are entirely incorporated herein byreference for all purposes.

PCR generally involves the heating and cooling of a reaction mixturethat includes several key reagents and a nucleic acid (e.g., DNA)template. Non-limiting examples of reagents that, in addition to anucleic acid template, can be used for PCR include primers, apolymerase, deoxynucleoside triphosphates (dNTPs), buffer solution,divalent cations, and monovalent cations. In general, at least twodifferent primers per nucleic acid template can be included in thereaction mixture, wherein each primer is complementary to a portion of(e.g., the 3′ ends of) the nucleic acid template. The nucleic acidtemplate is replicated by a polymerase.

Non-limiting examples of DNA polymerases that can be useful in PCRinclude Taq polymerase, Pfu polymerase, Pwo polymerase, Tfl polymerase,rTth polymerase, Tli polymerase, Tma polymerase, and VentR polymerase,Kapa2g polymerase, KOD polymerase, HaqZ05 polymerase, Haqz05 polymerase,or combinations thereof.

dNTPs are nucleotides that include triphosphate groups and are generallythe building-blocks from which amplified DNA is synthesized.Non-limiting examples of dNTPs useful in PCR include deoxyadenosinetriphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidinetriphosphate (dCTP), and deoxythymidine triphosphate (dTTP).

A buffer solution can be generally used to provide a suitable chemicalenvironment (e.g., pH, ionic strength, etc.) for optimum activity andstability of the DNA polymerase and/or other dependent components in thereaction mixture. For example, buffers of Tris-hydrochloride can beuseful in PCR methods.

Divalent cations can also be required for DNA polymerase functionality,with non-limiting examples including magnesium ions (Mg²⁺) and manganese(Mn²⁺) ions. Monovalent cations, such as, for example, potassium ions(K⁺) can be included and can be useful in minimizing the production ofunwanted, non-specific amplification products.

In some embodiments, the reagents for a PCR reaction can be a componentof an assay designed to test a blood or other liquid sample for thepresence of an analyte. For example, chloride ions can be measured byany of the following protocols, and components of these assays can bepresent in a storage site: Colorimetric methods: chloride ions displacethiocyanate from mercuric thiocyanate. Free thiocyanate reacts withferric ions to form a colored complex—ferric thiocyanate, which ismeasured photometrically. Coulometric methods: passage of a constantdirect current between silver electrodes produces silver ions, whichreact with chloride, forming silver chloride. After all the chloridecombines with silver ions, free silver ions accumulate, causing anincrease in current across the electrodes and indicating the end pointto the reaction. Mercurimetric methods: chloride is titrated with astandard solution of mercuric ions and forms HgCl2 soluble complex. Theend point for the reaction is detected colorimetrically when excessmercury ions combine with an indicator dye, diphenylcarbazon, to form ablue color. Likewise, magnesium can be measured colorimetrically usingcalmagite, which turns a red-violet color upon reaction with magnesium;by a formazan dye test; emits at 600 nm upon reaction with magnesium orusing methylthymol blue, which binds with magnesium to form a bluecolored complex. Likewise, calcium can be detected by a colorimetrictechnique using O-Cresolphtalein, which turns a violet color uponreaction of O-Cresolphtalein complexone with calcium. Likewise,Bicarbonate can be tested bichromatically because bicarbonate (HCO3⁻)and phosphoenolpyruvate (PEP) are converted to oxaloacetate andphosphate in the reaction catalyzed by phosphoenolpyruvate carboxylase(PEPC). Malate dehydrogenase (MD) catalyzes the reduction ofoxaloacetate to malate with the concomitant oxidation of reducednicotinamide adenine dinucleotide (NADH). This oxidation of NADH resultsin a decrease in absorbance of the reaction mixture measuredbichromatically at 380/410 nm proportional to the Bicarbonate content ofthe sample. Blood urea nitrogen can be detected in a colorimetric testin which diacetyl, or fearon develops a yellow chromogen with urea andcan be quantified by photometry, or multiusing the enzyme urease, whichconverts urea to ammonia and carbonic acid, which can be assayed by,e.g., i) decrease in absorbance at 340 nm when the ammonia reacts withalpha-ketoglutaric acid, ii) measuring the rate of increase inconductivity of the solution in which urea is hydrolyzed. Likewise,creatinine can be measured colorimetrically, by treated the sample withalkaline picrate solution to yield a red complex. In addition, creatinecan be measured using a non-Jaffe reaction that measures ammoniagenerated when creatinine is hydrolyzed by creatinine iminohydrolase.Glucose can be measured in an assay in which blood is exposed to a fixedquantity of glucose oxidase for a finite period of time to estimateconcentration. After the specified time, excess blood is removed and thecolor is allowed to develop, which is used to estimate glucoseconcentration. For example, glucose oxidase reaction with glucose formsnascent oxygen, which converts potassium iodide (in the filter paper) toiodine, forming a brown color. The concentration of glycosylatedhemoglobin as an indirect read of the level of glucose in the blood.When hemolysates of red cells are chromatographed, three or more smallpeaks named hemoglobin A1a, A1b, and A1c are eluted before the mainhemoglobin A peak. These “fast” hemoglobins are formed by theirreversible attachment of glucose to the hemoglobin in a two-stepreaction. Hexokinase can be measured in an assay in which glucose isphosphorylated by hexokinase (HK) in the presence of adenosinetriphosphate (ATP) and magnesium ions to produce glucose-6-phosphate andadenosine diphosphate (ADP). Glucose-6-phosphate dehydrogenase (G6P-DH)specifically oxidises glucose-6-phosphate to gluconate-6-phosphate withthe concurrent reduction of NAD+ to NADH. The increase in absorbance at340 nm is proportional to the glucose concentration in the sample. HDL,LDL, triglycerides can be measured using the Abell-Kendall protocol thatinvolves color development with Liebermann-Burchard reagent (mixedreagent of acetic anhydride, glacial acetic acid, and concentratedsulfuric acid) at 620 nm after hydrolysis and extraction of cholesterol.A fluorometric analysis can be used utilized to determine triglyceridereference values. Plasma high-density lipoprotein cholesterol (HDL-C)determination is measured by the same procedures used for plasma totalcholesterol, after precipitation of apoprotein B-containing lipoproteinsin whole plasma (LDL and VLDL) by heparin-manganese chloride. Thesecompounds can also be detected colorimetrically in an assay that isbased on the enzyme driven reaction that quantifies both cholesterolesters and free cholesterol. Cholesterol esters are hydrolyzed viacholesterol esterase into cholesterol, which is then oxidized bycholesterol oxidase into the ketone cholest-4-en-3-one plus hydrogenperoxide. The hydrogen peroxide is then detected with a highly specificcolorimetric probe. Horseradish peroxidase catalyzes the reactionbetween the probe and hydrogen peroxide, which bind in a 1:1 ratio.Samples can be compared to a known concentration of cholesterolstandard.

A single cycle of PCR typically comprises a series of steps that includea denaturation step, an annealing step, and an elongation step. Duringdenaturation, a double-stranded DNA template can be melted into itsindividual strands, such that the hydrogen bonds formed between bases ineach base-pair of the double-stranded DNA are broken.

After denaturation, an annealing step is completed, wherein the reactionmixture is incubated under conditions at which the primers hybridizewith complementary sequences present on each of the original individualstrands. After annealing, the elongation step commences, wherein theprimers are extended by a DNA polymerase, using dNTPs present in thereaction mixture. At the conclusion of elongation, two newdouble-stranded DNA molecules result, each comprising one of theoriginal individual strands of the DNA template. Each step of PCR isgenerally initiated by a change in the temperature of the reactionmixture that results from the heating or cooling of the reactionmixture. At the completion of a single round of amplification, thethermal cycle can be repeated for further rounds of amplification. Thegeneration of replicate amplification products is theoreticallyexponential with each subsequent thermal cycle. For example, for asingle DNA template, each step n, can result in a total of r replicates.

Successful PCR amplification requires high yield, high selectivity, anda controlled reaction rate at each step. Yield, selectivity, andreaction rate also generally depend on temperature, and optimaltemperatures depend on the composition and length of the polynucleotide,enzymes, and other components in the reaction mixture. In addition,different temperatures can be optimal for different steps or differentnucleic acids to be amplified. Moreover, optimal reaction conditions canvary, depending on the sequence of the template DNA, sequence of adesigned primer, and composition of the reaction mixture. Thermal cydersthat can be used to perform a PCR reaction can be programmed byselecting temperatures to be maintained, time durations for each portionof a cycle, number of cycles, rate of temperature change, and the like.

Primers for PCR can be designed according to known algorithms. Forexample, algorithms implemented in commercially available or customsoftware can be used to design primers. In some examples, primers canconsist of at least about 12 bases. In other examples, a primer canconsist of at least about 15, 18, or 20 bases in length. In still otherexamples, a primer can be up to 50+ bases in length. Primers can bedesigned such that all of the primers participating in a particularreaction have melting temperatures that are within at least about 5° C.,and more typically within about 2° C. of each other. Primers can befurther designed to avoid selfhybridization or hybridization with otherdesired primers. Those of skill in the art will recognize that theamount or concentration of primer in a reaction mixture will vary, forexample, according to the binding affinity of the primers for a giventemplate DNA and/or the quantity of available template DNA. Typicalprimer concentrations, for example, can range from 0.01 μM to 0.5 μM.

In an example PCR reaction, a reaction mixture, including adouble-stranded DNA template and additional reagents necessary for PCR,is heated to about 80-98° C. and held at that temperature for about10-90 seconds, in order to denature the DNA template into its individualstrands. Each individual strand, during the annealing step, is thenhybridized to its respective primer included in the reaction mixture bycooling the reaction mixture to a temperature of about 30-65° C. andholding it at that temperature for about 1-2 minutes. The elongationstep then commences, wherein elongation of the respective primershybridized to each individual strand occurs by the action of a DNApolymerase adding dNTPs to the primers. Elongation is initiated byheating the reaction mixture to a temperature of about 70-75° C. andholding at that temperature for 30 seconds to 5 minutes. The reactioncan be repeated for any desired number of cycles depending on, forexample, the initial amount of DNA template, the length of the desiredamplification product, the amount of dNTPs, the amount of primer, and/orprimer stringency.

While general PCR methods can be useful for nucleic acid amplification,other more specialized forms of PCR can be even more useful for a givenapplication. Nonlimiting examples of commonly used, more-specializedforms of PCR include reverse transcription PCR (RT-PCR) (e.g., U.S. Pat.No. 7,883,871), quantitative PCR (qPCR) (e.g., U.S. Pat. No. 6,180,349),real-time quantitative PCR (RTQ-PCR) (e.g., U.S. Pat. No. 8,058,054),allele-specific PCR (e.g., U.S. Pat. No. 5,595,890), assembly PCR (e.g.U.S. Patent Publication No. 20120178129), asymmetric PCR (e.g., EuropeanPatent Publication No. EP23 73 807), dial-out PCR (e.g., Schwartz J,NATURE METHODS, September 2012; 9(9): 913-915), helicase-dependent PCR(e.g., Vincent M, EMBO REPORTS 5, 2004, 5(8): 795-800), hot start PCR(e.g., European Patent Publication No. EP1419275), inverse PCR (e.g.,U.S. Pat. No. 6,607,899), methylation-specific PCR (e.g., EuropeanPatent Publication No. EP1690948), miniprimer PCR (U.S. PatentPublication No. 20120264132), multiplex PCR (U.S. Patent Publication No.20120264132), nested PCR (U.S. Patent Publication No. 20120264132),overlap-extension PCR (U.S. Patent Publication No. 20120264132), thermalasymmetric interlaced PCR (U.S. Patent Publication No. 20120264132), andtouchdown PCR (U.S. Patent Publication No. 20120264132). The device,apparatus, system, and/or method herein disclosed can be utilized toconduct such more-specialized forms of PCR.

RT-PCR Reactions

The device, apparatus, system, and method of the current disclosure caninclude the completion of an RT-PCR amplification reaction, and, thus, asample can comprise reagents necessary to complete a RT-PCR reaction.Non-limiting examples of such reagents include the reagents necessary tocomplete a PCR reaction, a reverse transcriptase, and a RNA templatethat can be used to synthesize a complementary DNA (cDNA) complement. Incases where reverse transcriptase must be removed prior to cDNAamplification, a sample supplied to a thermal cycler cannot containreagents necessary to complete a PCR reaction and can require a separateamplification reaction. Generally, the ratio of each reagent in thesample can vary and depend upon, for example, the amount of nucleic acidto be amplified and/or the desired amount of amplification products.Methods to determine the ratio of each reagent necessary for an RT-PCRamplification reaction are generally known by those skilled in the art.

Reverse transcription refers to a process by which ribonucleic acid(RNA) is replicated to its single-stranded complementary DNA (cDNA) by areverse transcriptase enzyme. Non-limiting examples of reversetranscriptase enzymes include Moloney murine leukemia virus (MMLV)transcriptase, avian myeloblastosis virus (AMY) transcriptase, variantsof AMV-transcriptase, or reverse transcriptases that have endo Hactivity. In reverse transcription PCR(RT-PCR), a reverse transcriptase,generally with endo H3 activity, is added to a reaction mixture thatincludes an RNA template and necessary reagents for PCR. The reversetranscriptase can complete RNA template replication to cDNA, byhybridizing dNTPs to the RNA template at proper conditions.

At the conclusion of replication, the reverse transcriptase can removethe single-stranded, cDNA replicated from the RNA template to permitadditional replication of the cDNA with PCR methods described above. ThecDNA and its amplification products that are produced from PCR can beused indirectly to garner information about the RNA, such as, forexample, the sequence of the RNA. The cDNA product that is synthesizedfrom an RNA by a reverse transcriptase can be removed from the reactionmixture to be used as a DNA template in a separate, subsequent set ofPCR reactions or amplification via PCR can occur in situ where reversetranscriptase is included in the reaction mixture with reagentsnecessary for PCR.

Q-PCR or RTQ-PCR Reactions

The device, apparatus, system, and method of the current disclosure caninclude the completion of a Q-PCR or RTQ-PCR amplification reaction,and, thus a sample can comprise reagents necessary to complete a Q-PCRor RTQ-PCR amplification reaction. Non-limiting examples of suchreagents include the reagents necessary to complete a PCR reaction and areporter used to detect amplification products. Generally, the ratio ofeach reagent in the sample can vary and depend upon, for example, theamount of nucleic acid to be amplified and/or the desired amount ofamplification products. Methods to determine the ratio of each reagentnecessary for a Q-PCR or RTQ-PCR amplification reaction are generallyknown by those skilled in the art.

Quantitative PCR (Q-PCR) is a variation of PCR in which the amount oftemplate DNA in a sample is quantified. Generally, amplificationproducts produced by PCR methods are linked to a reporter, such as, forexample, a fluorescent dye. At the end of a reaction, the reporter canbe detected and the results back-calculated (based on the associationratio of reporter to DNA and the known number of thermal cycles) todetermine the amount of original DNA template present. In some examples,the fluorescent dye can be detected in real time as amplificationprogresses. Such a variation of Q-PCR can be appropriately calledreal-time quantitative PCR (RTQ-PCR), real-time PCR, or kinetic PCR.Both Q-PCR and RTQ-PCR can be used to determine whether or not aspecific DNA template is present in a sample. In general, due to thepossible changes to reaction efficiency as the number of PCR cyclesincreases, however, RTQ-PCR methods can be generally more sensitive,more reliable, and thus, more frequently employed by those skilled inthe art as measurements are made on amplification products as they aresynthesized rather than on the aggregate of amplification productsobtained at the completion of the desired number of thermal cycles.Q-PCR and RTQ-PCR can also be combined with other PCR methods, such as,for example, RT-PCR. As an example, utility of combining Q-PCR orRTQ-PCR with other PCR methods, reporters can be included in an RT-PCRreaction mixture to detect and/or quantify low levels of messenger RNA(mRNA) via replication of its associated cDNA, which can enable thequantification of relative gene expression in a particular cell ortissue.

One or more reporters can be used to quantify DNA amplified as part ofQ-PCR and RTQ-PCR methods. Reporters can be associated with DNA both bycovalent and/or non-covalent linkages (e.g., ionic interactions, Van derWaals forces, hydrophobic interactions, hydrogen bonding, etc.). Forexample, a fluorescent dye that non-covalently intercalates withdouble-stranded DNA can be used as a reporter. In another example, a DNAoligonucleotide probe that fluoresces when hybridized with acomplementary DNA can be used as a reporter. In some examples, reporterscan bind to initial reactants and changes in reporter levels can be usedto detect amplified DNA. In other examples, reporters can only bedetectable or non-detectable as DNA amplification progresses. Detectionof reporters can be accomplished with one of many detection systems thatare suitable in the art. Optical detectors (e.g., fluorimeters,ultra-violet/visible light absorbance spectrophotometers) orspectroscopic detectors (e.g., nuclear magnetic resonance (NMR),infrared spectroscopy) can be, for example, useful modalities ofreporter detection. Gel based techniques, such as, for example, gelelectrophoresis can also be used for detection.

A reporter used in a Q-PCR or RTQ-PCR reaction can be an intercalatorthat can be detected. An intercalator generally binds to DNA bydisrupting hydrogen bonds between complementary bases, and, instead fitsitself between the disrupted bases. An intercalator can form its ownhydrogen bonds with one or more of the disrupted bases. Non-limitingexamples of intercalators include SYBR green, SYBR blue, DAPI, propidiumiodine, Hoeste, SYBR gold, ethidium bromide, acridines, proflavine,acridine orange, acriflavine, fluorcoumanin, ellipticine, daunomycin,chloroquine, distamycin D, chromomycin, homidium, mithramycin, rutheniumpolypyridyls, anthramycin, phenanthridines and acridines, ethidiumbromide, propidium iodide, hexidium iodide, dihydroethidium, ethidiumhomodimer-1 and -2, ethidium monoazide, and ACMA.

A reporter used in a Q-PCR or RTQ-PCR reaction can be a minor groovebinder that can be detected. Nonlimiting examples of minor grove bindersinclude indoles and imidazoles (e.g., Hoechst 33258, Hoechst 33342,Hoechst 34580 and DAPI).

A reporter used in a Q-PCR or RTQ-PCR reaction can be a nucleic acidstain that can be detected. Non-limiting examples of nucleic acid stainsinclude acridine orange (also capable of intercalating), 7-AAD,actinomycin D, LDS751, hydroxystilbamidine, SYTOX Blue, SYTOX Green,SYTOX Orange, POPO-1, POPO-3, YOYO-1, YOYO-3, TOTO-I, TOTO-3, JOJO-I,LOLO-1, BOBO-1, BOBO-3, PO-PRO-1, PO-PRO-3, BO-PRO-1, BO-PRO-3,TO-PRO-1, TO-PRO-3, TO-PRO-5, JO-PRO-1, LO-PRO-1, YO-PRO-1, YO-PRO-3,PicoGreen, OliGreen, RiboGreen, SYBR Gold, SYBR Green I, SYBR Green II,SYBR DX, SYTO-40, -41, -42, -43, -44, -45 (blue), SYTO-13, -16, -24,-21, -23, -12, -11, -20, -22, -15, -14, -25 (green), SYTO-81, -80, -82,-83, -84, -85 (orange), SYTO-64, -17, -59, -61, -62, -60, -63 (red).

A reporter used in a Q-PCR or RTQ-PCR reaction can be a fluorescent dyethat can be detected. Non-limiting examples of fluorescent dyes includefluorescein, fluorescein isothiocyanate (FITC), tetramethyl rhodamineisothiocyanate (TRITC), rhodamine, tetramethyl rhodamine,R-phycoerythrin, Cy-2, Cy-3, Cy-3.5, Cy-5, Cy5.5, Cy-7, Texas Red,Phar-Red, allophycocyanin (APC), Sybr Green I, Sybr Green II, Sybr Gold,CellTracker Green, 7-AAD, ethidium homodimer I, ethidium homodimer II,ethidium homodimer III, ethidium bromide, umbelliferone, eosin, greenfluorescent protein, erythrosin, coumarin, methyl coumarin, pyrene,malachite green, stilbene, lucifer yellow, cascade blue,dichlorotriazinylamine fluorescein, dansyl chloride, fluorescentlanthanide complexes such as those including europium and terbium,carboxy tetrachloro fluorescein, 5 and/or 6-carboxy fluorescein (FAM),5-(or 6-) iodoacetamidofluorescein, 5-{[2(and3)-5-(Acetylmercapto)-succinyl]amino} fluorescein (SAMSA-fluorescein),lissamine rhodamine B sulfonyl chloride, 5 and/or 6 carboxy rhodamine(ROX), 7-aminomethyl-coumarin, 7-Amino-4-methylcoumarin-3-acetic acid(AMCA), BODIPY fluorophores, 8-methoxypyrene-1,3,6-trisulfonic acidtrisodium salt, 3,6-Disulfonate-4-aminonaphthalimide, phycobiliproteins,AlexaFluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 633, 635,647, 660, 680, 700, 750, and 790 dyes, DyLight 350, 405, 488, 550, 594,633, 650, 680, 755, and 800 dyes, or other fluorophores known to thoseof skill in the art. For detailed listing of fluorophores that can beuseful in Q-PCR and RTQ-PCR methods, see also Hermanson, G. T.,BIOCONJUGATE TECHNIQUES (Academic Press, San Diego, 1996) and Lakowicz,J. R., PRINCIPLES OF FLUORESCENCE SPECTROSCOPY, (Plenum Pub Corp, 2ndedition (July 1999)), which are incorporated herein by reference.

A reporter used in a Q-PCR or RTQ-PCR reaction can be a radioactivespecies that can be detected. Nonlimiting examples of radioactivespecies that can be useful in Q-PCR and RTQ-PCR methods include 14C 12311241 1251 131 I, Tc99m, 35S, or 3H.

A reporter used in a Q-PCR or RTQ-PCR reaction can be an enzyme that canproduce a detectable signal. Such signal can be produced by action ofthe enzyme on its given substrate. Non-limiting examples of enzymes thatcan be useful in Q-PCR or RTQ-PCR methods include alkaline phosphatase,horseradish peroxidase, I2-galactosidase, alkaline phosphatase,galactosidase, acetylcholinesterase, and luciferase.

A reporter used in a Q-PCR or RTQ-PCR reaction can be an affinityligand-label that can be detected. A particular ligand can include alabel, such as for example, a fluorescent dye, and binding of thelabeled ligand to its substrate can produce a useful signal.Non-limiting examples of binding pairs that can be useful in Q-PCR orRTQ-PCR methods include streptavidin/biotin, avidin/biotin or anantigen/antibody complex, such as, for example, rabbit IgG andanti-rabbit IgG;

A reporter used in a Q-PCR or RTQ-PCR reaction can be a nanoparticlethat can be detected via light scattering or surface plasmon resonance(SPR). Non-limiting examples of materials useful for SPR-based detectioninclude gold and silver materials. Other nanoparticles that can beuseful in Q-PCR or RTQ-PCR reactions can be quantum dots (Qdots). Qdotsare generally constructed of semiconductor nanocrystals, described, forexample in U.S. Pat. No. 6,207,392. Nonlimiting examples ofsemiconductor materials that can be used to produce a Qdot include MgS,MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, ZnS,ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe, HgTe, GaAs, InGaAs, InP, InAs,or mixed compositions thereof.

A reporter used in a Q-PCR or RTQ-PCR reaction can be a labeledoligonucleotide probe. Probe based quantitative methods rely on thesequence-specific detection of amplification products of a desired DNAtemplate, using a labeled oligonucleotide. The oligonucleotide can be aprimer or a longer, different type of oligonucleotide. Theoligonucleotide can be DNA or RNA. As a result, unlike non-sequencespecific reporters, a labeled, sequence-specific probe hybridizes withseveral bases in an amplification product, and, thus, results inincreased specificity and sensitivity of detection. A label linked to aprobe can be any of the various reporters mentioned above and can alsoinclude a quencher (a molecule used, for example, to inhibitfluorescence). Methods for performing probe-based quantitativeamplification are described in U.S. Pat. No. 5,210,015, which isentirely incorporated herein by reference. Non-limiting examples ofprobes that can be useful in Q-PCR or RTQ-PCR reactions include TaqManprobes, TaqMan Tamara probes, TaqMan MGB probes, or Lion probes.

A variety of arrangements of quencher and fluorescent dye can be usedwhen both are used. In the case of a molecular beacon, for example, aquencher is linked to one end of an oligonucleotide capable of forming ahairpin structure. At the other end of the oligonucleotide is afluorescent dye. Unbound to a complementary sequence on an amplificationproduct, the oligonucleotide inter-hybridizes with itself and assumes ahairpin configuration. In the hairpin configuration, the fluorescent dyeand quencher are brought in close proximity which effectively preventsfluorescence of the dye. Upon hybridizing with an amplification productof a desired template DNA, however, the oligonucleotide hybridizes in alinear fashion, the fluorescence and quencher separate, and fluorescencefrom the dye can be achieved and subsequently detected. In otherexample, a linear, RNA based probe that includes a fluorescent dye and aquencher held in adjacent positions can be used for detection. The closeproximity of the dye to the quencher prevents its fluorescence. Upon thebreakdown of the probe with the exonuclease activity of a DNApolymerase, however, the quencher and dye are separated, and the freedye can fluoresce and be detected. As different probes can be designedfor different sequences, multiplexing is possible. In a multiplexeddetection, assaying for several DNA templates in the same reactionmixture can be possible by using different probes, each labeled with adifferent reporter, for each desired DNA template.

A Q-PCR or RTQ-PCR reaction can include a single reporter or can includemultiple reporters. One or more detection methodologies can be used forquantification. Moreover, as Q-PCR and RT-PCR generally adds just aquantification step, it can be generally linked to any type of PCRreaction.

LCR Reactions

The device, apparatus, system, and method of the current disclosure caninclude the completion of a LCR amplification reaction (or any step of aLCR reaction-as described elsewhere herein), and, thus, a sample cancomprise reagents necessary to complete a LCR amplification reaction.Non-limiting examples of such reagents include a template DNA moleculeto be amplified, a set of oligonucleotide probes that can each hybridizewith a different, but adjacent to the other, portion of a targetsequence on the template DNA, a DNA ligase, a buffer at a pH andconcentration suitable for a desired LCR reaction, a monovalent cation,and a divalent cation. Generally, the ratio of each reagent in thesample can vary and depend upon, for example, the amount of nucleic acidto be amplified and/or the desired amount of amplification products.Methods to determine the ratio of each reagent necessary for a LCRamplification reaction are generally known by those skilled in the art.

LCR is generally a method similar to PCR, with some important keydistinctions. A key distinction of general LCR over PCR, is that LCRamplifies an oligonucleotide probe using a DNA ligase enzyme to produceamplification products instead of through polymerization of nucleotideswith a DNA polymerase. In LCR, two complementary oligonucleotide probepairs that are specific to a DNA template can be used. Afterdenaturation of a to-be-replicated template DNA into its individualstrands, each probe pair can hybridize to adjacent positions on itsrespective individual strand of the template. Primers are generally notused in LCR. Any gap and/or nick created by the joining of two probescan be sealed by the enzyme DNA ligase, in order to produce a continuousstrand of DNA complementary to the template DNA. Similar to PCR, though,LCR generally requires thermal cycling, with each part of the thermalcycle driving a particular step of the reaction. Repeated temperaturechanges can result in the denaturation of the DNA template, annealing ofthe oligonucleotide probes, ligation of the oligonucleotide probes, andseparation of the ligated unit from the original DNA template. Moreover,a ligated unit synthesized in one thermal cycle can be replicated in thenext thermal cycle. Each thermal cycle can result in a doubling of theDNA template, resulting in exponential amplification of the template DNAin a fashion analogous to PCR.

Gap LCR Reactions

The device, apparatus, system, and method of the current disclosure caninclude the completion of a gap LCR amplification reaction, and, thus, asample can comprise reagents necessary to complete a gap LCRamplification reaction. Non-limiting examples of such reagents includethe reagents necessary to complete a LCR reaction, wherein the set ofoligonucleotide probes can each hybridize with a different, non-adjacentportion of a target sequence on the template DNA, dNTPs, and a DNApolymerase. Generally, the ratio of each reagent in the sample can varyand depend upon, for example, the amount of nucleic acid to be amplifiedand/or the desired amount of amplification products. Methods todetermine the ratio of each reagent necessary for a gap LCRamplification reaction are generally known by those skilled in the art.

Gap LCR is a specialized type of LCR that utilizes modifiedoligonucleotide probes that cannot be ligated if a specific sequence isnot present on a DNA template. The probes can be designed in a way thatwhen they hybridize to an individual strand of a DNA template, they doso discontinuously and are generally separated by a gap of one toseveral base pairs. The gap can be filled by with dNTPs using a DNApolymerase, which can result in adjacency of the two original probes. Asin general LCR, DNA ligase can join the two resulting, adjacent probesin order to produce a continuous strand of DNA complementary to theoriginal template. The newly synthesized strand can then be used forfurther thermal cycles of template amplification. Gap LCR generally hashigher sensitivity than LCR as it minimizes ligation where a desiredsequence is not present on a template DNA. Moreover, the combined use ofboth DNA ligase and DNA polymerase can also result in a more accurateidentification of a sequence of interest, even in cases where low levelsof DNA template are available.

Additionally, since LCR is a DNA replication method, analogous methodsto RT-PCR, Q-PCR, and RTQPCR are possible. For example, any of thereporters specified above can be considered for use in a quantitative(Q-LCR) or real-time quantitative LCR (RTQ-LCR) reaction. Moreover, LCRmethods can be combined with PCR or other nucleic amplificationtechniques.

Q-LCR and LTQ-LCR Reactions

The device, apparatus, system, and method of the current disclosure caninclude the completion of a Q-LCR or LTQ-PCR reaction, and, thus, asample can comprise reagents necessary to complete a Q-LCR or RTQ-LCRreaction. Non-limiting examples of such reagents include the reagentsnecessary to complete a LCR reaction and a reporter used to detectamplification products. Generally, the ratio of each reagent in thesample can vary and depend upon, for example, the amount of nucleic acidto be amplified and/or the desired amount of amplification products.Methods to determine the ratio of each reagent necessary for a Q-LCR andRTQ-LCR amplification reaction are generally known by those skilled inthe art.

Since LCR is a DNA replication method, analogous methods to RT-PCR,Q-PCR, and RTQPCR are possible. For example, any of the reportersspecified above can be considered for use in a quantitative (Q-LCR) orreal-time quantitative LCR (RTQ-LCR) reaction. Moreover, LCR methods canbe combined with PCR or other nucleic amplification techniques.

Digital Nucleic Acid Amplification Reactions

The device, apparatus, system, and method of the current disclosure caninclude the completion of a digital nucleic acid amplification reaction,and, thus, a sample can comprise reagents necessary to complete adigital nucleic acid amplification reaction. In general, any of theexample nucleic acid amplification reactions discussed herein can beconducted in digital form, upon proper separation of a sample and/orreagents necessary for nucleic acid amplification into smallerpartitions. In some embodiments, such partitions can be droplets or canbe larger aliquots of the original sample. Generally, the ratio of eachreagent in partitions can vary and depend upon, for example, the amountof nucleic acid to be amplified in each droplet and/or the desiredamount of amplification products. Methods to determine the ratio of eachreagent necessary for a particular digital nucleic acid amplificationreaction are generally known by those skilled in the art.

Digital nucleic acid amplification is a technique that allowsamplification of a subset of nucleic acid templates fractioned intopartitions obtained from a larger sample. In some cases, a partition cancomprise a single nucleic acid template, such that amplificationproducts generated from amplification of the template are exclusivelyderived from the template. Amplification products can be detected usinga reporter, including any of those example reporters described herein.The amplification of a single nucleic acid template can be useful indiscriminating genetic variations that include, for example, wild-typealleles, mutant alleles, maternal alleles, or paternal alleles of agene. More comprehensive discussions of this technology, with respect toPCR, can be found elsewhere-see Pohl et al., Expert Rev. Mol. Diagn.,4(1):41-7 (2004), and Vogelstein and Kinzler, Proc. Natl. Acad. Sci. USA96:9236-9241 (1999), which are both incorporated herein in entirety byreference. So long as the proper thermal cycling of a partitioncomprising a complete reaction mixture (e.g., a reaction mixturecomprising both the nucleic acid template to be amplified and therequired reagents for the desired nucleic acid amplification reaction)is achieved, any of the example nucleic acid amplification reactionsdiscussed herein can be conducted digitally. Indeed, digital nucleicacid amplification methods still require thermal cycling and accuratetemperature control, as do their non-digital analogues.

In a digital nucleic acid amplification reaction, a large sample isfractioned into a number of smaller partitions, whereby the partitionscan contain on average a single copy of a nucleic acid template ormultiple copies of a template. Individual nucleic acid molecules can bepartitioned with the aid of a number of devices and strategies withnon-limiting examples that include micro-well plates, capillaries,dispersions that comprise emulsions, arrays of miniaturized chambers,nucleic acid binding surfaces, flow cells, droplet partitioning, orcombinations thereof. Each partition can be thermal cycled to generateamplification products of its component template nucleic acid, using anucleic acid amplification reaction of choice with non-limiting examplesof such reactions that include a digital PCR (dPCR) nucleic acidamplification reaction, a digital LCR (dLCR) nucleic acid amplificationreaction, a digital RT-PCR (dRT-PCR) nucleic acid amplificationreaction, a digital (dRT-LCR) nucleic acid amplification reaction, adigital Q-PCR (dQ-PCR) nucleic acid amplification reaction, a digitalQ-LCR (dQ-LCR) nucleic acid amplification reaction, a digital RTQ-PCR(dRTQ-PCR) nucleic acid amplification reaction, a digital LTQ-LCR(dLTQ-LCR) nucleic acid amplification reaction, or combinations thereof.

In cases where reporters are used, each partition can be considered“positive” or “negative” for a particular nucleic acid template ofinterest. The number of positives can be counted and, thus, one candeduce the starting amount of the template in the pre-partitioned samplebased upon the count. In some examples, counting can be achieved byassuming that the partitioning of the nucleic acid template populationin the original sample follows a Poisson distribution. Based on such ananalysis, each partition is labeled as either containing a nucleic acidtemplate of interest (e.g., labeled “positive”) or not containing thenucleic acid template of interest (e.g., labeled “negative”). Afternucleic acid amplification, templates can be quantified by counting thenumber of partitions that comprise “positive” reactions. Moreover,digital nucleic acid amplification is not dependent on the number ofamplification cycles to determine the initial amount of nucleic acidtemplate present in the original sample. This lack of dependencyeliminates relying on assumptions with respect to uncertain exponentialamplification, and, therefore, provides a method of direct, absolutequantification.

Most commonly, multiple serial dilutions of a starting sample are usedto arrive at the proper concentration of nucleic acid templates in thepartitions. The volume of each partition can depend on a host of factorsthat include, for example, the volume capacity of a thermal cycler usedto generate amplification products. Furthermore, quantitative analysesconducted by digital nucleic acid amplification can generally requirereliable amplification of single copies of nucleic acid template withlow false positive rates. Such capability can require carefuloptimization in microliter-scale vessels. Moreover, the analyticalprecision of a nucleic acid amplification reaction can be dependent onthe number of reactions.

In some embodiments, digital nucleic acid amplification reactions can bedroplet digital nucleic acid amplification reactions. Non-limitingexamples of such nucleic acid amplification reactions include dropletdigital PCR (ddPCR), droplet digital RT-PCR (ddRT-PCR), droplet digitalQ-PCR (ddQ-PCR), droplet digital RTQ-PCR (ddRTQ-PCR), droplet digitalLCR (ddLCR), droplet digital RT-LCR (ddRT-LCR), droplet digital Q-LCR(ddQ-LCR), or droplet digital RTQ-LCR (ddRTQ-PCR), or combinationsthereof.

In some cases, a digital nucleic acid amplification reaction can be adroplet digital nucleic acid amplification reaction. For example, such anucleic acid amplification reaction can be a droplet digital PCR (ddPCR)nucleic acid amplification reaction. A ddPCR nucleic acid amplificationreaction can be completed by first partitioning a larger samplecomprising nucleic acids into a plurality of droplets. Each dropletcomprises a random partition of nucleic acids in the original sample.The droplets can then be combined with different droplets that comprisethe reagents necessary for a PCR reaction (e.g., a set of two primersthat can hybridize with a target sequence on the template DNA, a DNApolymerase, deoxynucleotide triphosphates (dNTPs), a buffer at a pH andconcentration suitable for a desired PCR reaction, a monovalent cation,and a divalent cation). The new combined droplet is then properlythermal cycled in a thermal cycler and PCR commences. Alternatively, asample can already comprise reagents necessary for PCR prior topartitioning into droplets—droplet combination with other dropletswould, thus, not be required.

Analogous procedures can be followed to complete a droplet digitalRT-PCR (ddRT-PCR) nucleic acid amplification reaction, a droplet digitalLCR (ddLCR) nucleic acid amplification reaction, a droplet digitalRT-PCR (ddRT-LCR) nucleic acid amplification reaction, a droplet digitalQ-PCR (ddQ-PCR) nucleic acid amplification reaction, a droplet digitalRTQ-PCR (ddRTQ-PCR) nucleic acid amplification reaction, a dropletdigital Q-LCR (ddQ-LCR) nucleic acid amplification reaction, or adroplet digital RTQ-LCR (ddRTQ-LCR) reaction.

In the case of a quantitative droplet digital nucleic acid amplificationreaction (e.g., ddQ-PCR, ddRTQ-PCR, ddQ-LCR, or ddRTQ-LCR), droplets canalso comprise a reporter used to detect amplification products. Suchreporters can be contacted with nucleic acids by combining droplets orcan already be included in a partition comprising nucleic acid templatesto be amplified.

Droplet nucleic acid amplification can be completed using a variety ofsample holders. In some examples, droplets can be applied to one or morewells of a sample holder and then thermal cycled. In other examples, adevice comprising fluidic channels, such as, for example, a flow cell ormicrofluidic device can be used. Fluidic channels can be used totransport droplets through a sample holder (or other component of athermal cycler) such that droplet thermal contact with differenttemperature regions of the sample holder (or other component of athermal cycler) results in proper thermal cycling of the droplets.

Related Documents

The present invention includes a variety of embodiments, which can becombined in multiple ways as long as the various components do notcontradict one another. The embodiments should be regarded as a singleinvention file: each filing has other filing as the references and isalso referenced in its entirety and for all purpose, rather than as adiscrete independent. These embodiments include not only the disclosuresin the current file, but also the documents that are herein referenced,incorporated, or to which priority is claimed.

Definitions

The terms used in describing the devices, systems, and methods hereindisclosed are defined in the current application, or in PCT Application(designating U.S.) Nos. PCT/US2016/045437 and PCT/US0216/051775, whichwere respectively filed on Aug. 10, 2016 and Sep. 14, 2016, U.S.Provisional Application No. 62/456,065, which was filed on Feb. 7, 2017,U.S. Provisional Application No. 62/456,287, which was filed on Feb. 8,2017, and U.S. Provisional Application No. 62/456,504, which was filedon Feb. 8, 2017, all of which applications are incorporated herein intheir entireties for all purposes.

The terms “CROF Card (or card)”, “COF Card”, “QMAX-Card”, “Q-Card”,“CROF device”, “COF device”, “QMAX-device”, “CROF plates”, “COF plates”,and “QMAX-plates” are interchangeable, except that in some embodiments,the COF card does not comprise spacers; and the terms refer to a devicethat comprises a first plate and a second plate that are movablerelative to each other into different configurations (including an openconfiguration and a closed configuration), and that comprises spacers(except some embodiments of the COF card) that regulate the spacingbetween the plates. The term “X-plate” refers to one of the two platesin a CROF card, wherein the spacers are fixed to this plate. Moredescriptions of the COF Card, CROF Card, and X-plate are given in theprovisional application Ser. No. 62/456,065, filed on Feb. 7, 2017,which is incorporated herein in its entirety for all purposes.

A RHC card (a sample holder) includes a Q-card.

(2) Q-Card, Spacer and Uniform Sample Thickness

The devices, systems, and methods herein disclosed can include or useQ-cards, spacers, and uniform sample thickness embodiments for sampledetection, analysis, and quantification. In some embodiments, the Q-cardcomprises spacers, which help to render at least part of the sample intoa layer of high uniformity. The structure, material, function, variationand dimension of the spacers, as well as the uniformity of the spacersand the sample layer, are herein disclosed, or listed, described, andsummarized in PCT Application (designating U.S.) Nos. PCT/US2016/045437and PCT/US0216/051775, which were respectively filed on Aug. 10, 2016and Sep. 14, 2016, U.S. Provisional Application No. 62/456,065, whichwas filed on Feb. 7, 2017, U.S. Provisional Application No. 62/456,287,which was filed on Feb. 8, 2017, and U.S. Provisional Application No.62/456,504, which was filed on Feb. 8, 2017, all of which applicationsare incorporated herein in their entireties for all purposes.

(3) Hinges, Opening Notches, Recessed Edge and Sliders

The devices, systems, and methods herein disclosed can include or useQ-cards for sample detection, analysis, and quantification. In someembodiments, the Q-card comprises hinges, notches, recesses, andsliders, which help to facilitate the manipulation of the Q card and themeasurement of the samples. The structure, material, function, variationand dimension of the hinges, notches, recesses, and sliders are hereindisclosed, or listed, described, and summarized in PCT Application(designating U.S.) Nos. PCT/US2016/045437 and PCT/US0216/051775, whichwere respectively filed on Aug. 10, 2016 and Sep. 14, 2016, U.S.Provisional Application No. 62/456,065, which was filed on Feb. 7, 2017,U.S. Provisional Application No. 62/456,287, which was filed on Feb. 8,2017, and U.S. Provisional Application No. 62/456,504, which was filedon Feb. 8, 2017, all of which applications are incorporated herein intheir entireties for all purposes.

(4) Q-Card, Sliders, and Smartphone Detection System

The devices, systems, and methods herein disclosed can include or useQ-cards for sample detection, analysis, and quantification. In someembodiments, the Q-cards are used together with sliders that allow thecard to be read by a smartphone detection system. The structure,material, function, variation, dimension and connection of the Q-card,the sliders, and the smartphone detection system are herein disclosed,or listed, described, and summarized in PCT Application (designatingU.S.) Nos. PCT/US2016/045437 and PCT/US0216/051775, which wererespectively filed on Aug. 10, 2016 and Sep. 14, 2016, U.S. ProvisionalApplication No. 62/456,065, which was filed on Feb. 7, 2017, U.S.Provisional Application No. 62/456,287, which was filed on Feb. 8, 2017,and U.S. Provisional Application No. 62/456,504, which was filed on Feb.8, 2017, all of which applications are incorporated herein in theirentireties for all purposes.

(5) Detection Methods

The devices, systems, and methods herein disclosed can include or beused in various types of detection methods. The detection methods areherein disclosed, or listed, described, and summarized in PCTApplication (designating U.S.) Nos. PCT/US2016/045437 andPCT/US0216/051775, which were respectively filed on Aug. 10, 2016 andSep. 14, 2016, U.S. Provisional Application No. 62/456,065, which wasfiled on Feb. 7, 2017, U.S. Provisional Application No. 62/456,287,which was filed on Feb. 8, 2017, and U.S. Provisional Application No.62/456,504, which was filed on Feb. 8, 2017, all of which applicationsare incorporated herein in their entireties for all purposes.

(6) Labels

The devices, systems, and methods herein disclosed can employ varioustypes of labels that are used for analytes detection. The labels areherein disclosed, or listed, described, and summarized in PCTApplication (designating U.S.) Nos. PCT/US2016/045437 andPCT/US0216/051775, which were respectively filed on Aug. 10, 2016 andSep. 14, 2016, U.S. Provisional Application No. 62/456,065, which wasfiled on Feb. 7, 2017, U.S. Provisional Application No. 62/456,287,which was filed on Feb. 8, 2017, and U.S. Provisional Application No.62/456,504, which was filed on Feb. 8, 2017, all of which applicationsare incorporated herein in their entireties for all purposes.

In some embodiments, labeling an analyte includes using, for example, alabeling agent, such as an analyte specific binding member that includesa detectable label. Detectable labels include, but are not limited to,fluorescent labels, colorimetric labels, chemiluminescent labels,enzyme-linked reagents, multicolor reagents, avidin-streptavidinassociated detection reagents, and the like. In some embodiments, thedetectable label is a fluorescent label. Fluorescent labels are labelingmoieties that are detectable by a fluorescence detector. For example,binding of a fluorescent label to an analyte of interest allows theanalyte of interest to be detected by a fluorescence detector. Examplesof fluorescent labels include, but are not limited to, fluorescentmolecules that fluoresce upon contact with a reagent, fluorescentmolecules that fluoresce when irradiated with electromagnetic radiation(e.g., UV, visible light, x-rays, etc.), and the like.

In some embodiments, suitable fluorescent molecules (fluorophores) forlabeling include, but are not limited to, IRDye800CW, Alexa 790, Dylight800, fluorescein, fluorescein isothiocyanate, succinimidyl esters ofcarboxyfluorescein, succinimidyl esters of fluorescein, 5-isomer offluorescein dichlorotriazine, cagedcarboxyfluorescein-alanine-carboxamide, Oregon Green 488, Oregon Green514; Lucifer Yellow, acridine Orange, rhodamine, tetramethylrhodamine,Texas Red, propidium iodide, JC-1(5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazoylcarbocyanineiodide), tetrabromorhodamine 123, rhodamine 6G, TMRM (tetramethylrhodamine methyl ester), TMRE (tetramethyl rhodamine ethyl ester),tetramethylrosamine, rhodamine B and 4-dimethylaminotetramethylrosamine,green fluorescent protein, blue-shifted green fluorescent protein,cyan-shifted green fluorescent protein, red-shifted green fluorescentprotein, yellow-shifted green fluorescent protein,4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid; acridine andderivatives, such as acridine, acridine isothiocyanate;5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS);4-amino-N-[3-vinylsulfonyl)phenyl]naphth-alimide-3,5 disulfonate;N-(4-anilino-1-naphthyl)maleimide; anthranilamide;4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a diaza-5-indacene-3-propionicacid BODIPY; cascade blue; Brilliant Yellow; coumarin and derivatives:coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin120),7-amino-4-trifluoromethylcoumarin (Coumarin 151); cyanine dyes;cyanosine; 4′,6-diaminidino-2-phenylindole (DAPI);5′,5″-dibromopyrogallolsulfonaphthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriaamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2-,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-(dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansylchloride);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives: eosin, eosin isothiocyanate, erythrosin and derivatives:erythrosin B, erythrosin, isothiocyanate; ethidium; fluorescein andderivatives: 5-carboxyfluorescein(FAM),5-(4,6-dichlorotriazin-2-yl)amino-fluorescein (DTAF),2′,7′dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), fluorescein,fluorescein isothiocyanate, QFITC, (XRITC); fluorescamine; IR144;IR1446; Malachite Green isothiocyanate; 4-methylumbelliferoneorthocresolphthalein; nitrotyrosine; pararosaniline; Phenol Red;B-phycoerythrin; ophthaldialdehyde; pyrene and derivatives: pyrene, 5pyrene butyrate, succinimidyl 1-pyrene; butyrate quantum dots; ReactiveRed 4 (Cibacron™ Brilliant Red 3B-A) rhodamine and derivatives:6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissaminerhodamine B sulfonyl chloride rhodamine (Rhod), rhodamine B, rhodamine123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101,sulfonyl chloride derivative of sulforhodamine 101 (Texas 10 Red);N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine;tetramethyl rhodamine isothiocyanate (TRITC); riboflavin;5-(2′-aminoethyl) aminonaphthalene-1-sulfonic acid (EDANS),4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL), rosolic acid; CALFluor Orange 560; terbium chelate derivatives; Cy 3; Cy 5; Cy 5.5; Cy 7;IRD 700; IRD 800; La Jolla Blue; phthalo cyanine; and naphthalo cyanine,coumarins and related dyes, xanthene dyes such as rhodols, resorufins,bimanes, acridines, isoindoles, dansyl dyes, aminophthalic hydrazidessuch as luminol, and isoluminol derivatives, aminophthalimides,aminonaphthalimides, aminobenzofurans, aminoquinolines,dicyanohydroquinones, fluorescent europium and terbium complexes;combinations thereof, and the like.

Suitable fluorescent proteins and chromogenic proteins include, but arenot limited to, a green fluorescent protein (GFP), including, but notlimited to, a GFP derived from Aequoria victoria or a derivativethereof, e.g., a “humanized” derivative such as Enhanced GFP; a GFP fromanother species such as Renilla reniformis, Renilla mulleri, orPtilosarcus guernyi; “humanized” recombinant GFP (hrGFP); any of avariety of fluorescent and colored proteins from Anthozoan species;combinations thereof; and the like.

In some embodiments, dyes that can be used to stain blood cells compriseWright's stain (Eosin, methylene blue), Giemsa stain (Eosin, methyleneblue, and Azure B), Can-Grünwald stain, Leishman's stain (“Polychromed”methylene blue (i.e. demethylated into various azures) and eosin),Erythrosine B stain (Erythrosin B), and other fluorescence stainincluding but not limit to Acridine orange dye,3,3-dihexyloxacarbocyanine (DiOC6), Propidium Iodide (PI), FluoresceinIsothiocyanate (FITC) and Basic Orange 21 (BO21) dye, Ethidium Bromide,Brilliant Sulfaflavine and a Stilbene Disulfonic Acid derivative,Erythrosine B or trypan blue, Hoechst 33342, Trihydrochloride,Trihydrate, and DAPI (4′,6-Diamidino-2-Phenylindole, Dihydrochloride).

In some embodiments, the labeling agent is configured to bindspecifically to the analyte of interest. In some embodiments, a labelingagent can be present in the device before the sample is applied to thedevice. In some embodiments, the device can be washed after the labelingagent is bound to the analyte-capture agent complex to remove from thedevice any excess labeling agent that is not bound to an analyte-captureagent complex.

In some embodiments, the analyte is labeled after the analyte is boundto the device, e.g., using a labeled binding agent that can bind to theanalyte simultaneously as the capture agent to which the analyte isbound in the CROF device, i.e., in a sandwich-type assay.

In some embodiments, a nucleic acid analyte can be captured on thedevice, and a labeled nucleic acid that can hybridize to the analytesimultaneously as the capture agent to which the nucleic acid analyte isbound in the device.

(7) Analytes

The devices, systems, and methods herein disclosed can be applied tomanipulation and detection of various types of analytes (includingbiomarkers). The analytes and are herein disclosed, or listed,described, and summarized in PCT Application (designating U.S.) Nos.PCT/US2016/045437 and PCT/US0216/051775, which were respectively filedon Aug. 10, 2016 and Sep. 14, 2016, U.S. Provisional Application No.62/456,065, which was filed on Feb. 7, 2017, U.S. ProvisionalApplication No. 62/456,287, which was filed on Feb. 8, 2017, and U.S.Provisional Application No. 62/456,504, which was filed on Feb. 8, 2017,all of which applications are incorporated herein in their entiretiesfor all purposes.

(8) Applications (Field and Samples)

The devices, systems, and methods herein disclosed can be used forvarious applications (fields and samples). The applications are hereindisclosed, or listed, described, and summarized in PCT Application(designating U.S.) Nos. PCT/US2016/045437 and PCT/US0216/051775, whichwere respectively filed on Aug. 10, 2016 and Sep. 14, 2016, U.S.Provisional Application No. 62/456,065, which was filed on Feb. 7, 2017,U.S. Provisional Application No. 62/456,287, which was filed on Feb. 8,2017, and U.S. Provisional Application No. 62/456,504, which was filedon Feb. 8, 2017, all of which applications are incorporated herein intheir entireties for all purposes.

(9) Cloud

The devices, systems, and methods herein disclosed can employ cloudtechnology for data transfer, storage, and/or analysis. The relatedcloud technologies are herein disclosed, or listed, described, andsummarized in PCT Application (designating U.S.) Nos. PCT/US2016/045437and PCT/US0216/051775, which were respectively filed on Aug. 10, 2016and Sep. 14, 2016, U.S. Provisional Application No. 62/456,065, whichwas filed on Feb. 7, 2017, U.S. Provisional Application No. 62/456,287,which was filed on Feb. 8, 2017, and U.S. Provisional Application No.62/456,504, which was filed on Feb. 8, 2017, all of which applicationsare incorporated herein in their entireties for all purposes.

Additional Notes

Further examples of inventive subject matter according to the presentdisclosure are described in the following enumerated paragraphs.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise, e.g., when the word “single” isused. For example, reference to “an analyte” includes a single analyteand multiple analytes, reference to “a capture agent” includes a singlecapture agent and multiple capture agents, reference to “a detectionagent” includes a single detection agent and multiple detection agents,and reference to “an agent” includes a single agent and multiple agents.

As used herein, the terms “adapted” and “configured” mean that theelement, component, or other subject matter is designed and/or intendedto perform a given function. Thus, the use of the terms “adapted” and“configured” should not be construed to mean that a given element,component, or other subject matter is simply “capable of” performing agiven function. Similarly, subject matter that is recited as beingconfigured to perform a particular function may additionally oralternatively be described as being operative to perform that function.

As used herein, the phrase, “for example,” the phrase, “as an example,”and/or simply the terms “example” and “exemplary” when used withreference to one or more components, features, details, structures,embodiments, and/or methods according to the present disclosure, areintended to convey that the described component, feature, detail,structure, embodiment, and/or method is an illustrative, non-exclusiveexample of components, features, details, structures, embodiments,and/or methods according to the present disclosure. Thus, the describedcomponent, feature, detail, structure, embodiment, and/or method is notintended to be limiting, required, or exclusive/exhaustive; and othercomponents, features, details, structures, embodiments, and/or methods,including structurally and/or functionally similar and/or equivalentcomponents, features, details, structures, embodiments, and/or methods,are also within the scope of the present disclosure.

As used herein, the phrases “at least one of” and “one or more of,” inreference to a list of more than one entity, means any one or more ofthe entity in the list of entity, and is not limited to at least one ofeach and every entity specifically listed within the list of entity. Forexample, “at least one of A and B” (or, equivalently, “at least one of Aor B,” or, equivalently, “at least one of A and/or B”) may refer to Aalone, B alone, or the combination of A and B.

As used herein, the term “and/or” placed between a first entity and asecond entity means one of (1) the first entity, (2) the second entity,and (3) the first entity and the second entity. Multiple entity listedwith “and/or” should be construed in the same manner, i.e., “one ormore” of the entity so conjoined. Other entity may optionally be presentother than the entity specifically identified by the “and/or” clause,whether related or unrelated to those entities specifically identified.

Where numerical ranges are mentioned herein, the invention includesembodiments in which the endpoints are included, embodiments in whichboth endpoints are excluded, and embodiments in which one endpoint isincluded and the other is excluded. It should be assumed that bothendpoints are included unless indicated otherwise. Furthermore, unlessotherwise indicated or otherwise evident from the context andunderstanding of one of ordinary skill in the art.

In the event that any patents, patent applications, or other referencesare incorporated by reference herein and (1) define a term in a mannerthat is inconsistent with and/or (2) are otherwise inconsistent with,either the non-incorporated portion of the present disclosure or any ofthe other incorporated references, the non-incorporated portion of thepresent disclosure shall control, and the term or incorporateddisclosure therein shall only control with respect to the reference inwhich the term is defined and/or the incorporated disclosure was presentoriginally.

Additional Exemplary Embodiments

A device, comprising:

a first plate comprising a polymer material and having a thickness lessthan or equal to 100 μm;

a second plate comprising a polymer material and having a thickness lessthan or equal to 100 μm; and

a heating/cooling layer disposed on either the first plate or the secondplate, the heating/cooling layer having a thermal conductivity between6×10⁻⁵ W/K multiplied by the thickness of the heating/cooling layer and1.5×10⁻⁴ W/K multiplied by the thickness of the heating/cooling layer,

wherein the first plate and the second plate face each other in aparallel arrangement, and are separated from each other by a distance,and wherein the first plate and the second plate are configured toreceive a fluid sample sandwiched between the first plate and the secondplate.

The device of any prior embodiment, further comprising a light absorbinglayer disposed on the heating/cooling layer, wherein the light absorbinglayer has an average light absorptance of at least 30%.

The device of any prior embodiment, wherein the light absorbing layercomprises black paint.

The device of any prior embodiment, wherein the first plate is movablerelative to the second plate.

The device of any prior embodiment, wherein a thickness of theheating/cooling layer is less than or equal to 3 μm.

The device of any prior embodiment, wherein at least one of the firstplate and the second plate has an area across its major surface of about400 mm².

The device of any prior embodiment, further comprising a plurality ofspherical spacers disposed between the first plate and the second plate.

The device of any prior embodiment, further comprising a plurality ofspacers having a height of about 10 um, wherein the plurality of spacersare disposed between the first plate and the second plate.

The device of any prior embodiment, wherein the distance between thefirst plate and the second plate is less than or equal to 100 μm. Thedevice of any prior embodiment, further comprising a hinge configured toconnect the first plate with the second plate, and coupled to an edge ofthe first plate or the second plate.

A device, comprising:

a first plate;

a second plate having a thickness less than or equal to 100 μm, whereinthe second plate is separated from the first plate in a parallelarrangement by a distance less than or equal to the thickness of thesecond plate; and

a heating/cooling layer disposed on either the first plate or the secondplate,

wherein the heating/cooling layer is configured to receiveelectromagnetic radiation such that at least a portion of a liquidsample sandwiched between the first plate and the second plate is heatedat a rate of at least 30° C./sec.

The device of any prior embodiment, further comprising a light absorbinglayer disposed on the heating/cooling layer, wherein the light absorbinglayer has an average light absorptance of at least 30%.

The device of any prior embodiment, wherein the light absorbing layercomprises black paint.

The device of any prior embodiment, wherein the first plate is movablerelative to the second plate.

The device of any prior embodiment, wherein a thickness of theheating/cooling layer is less than or equal to 3 μm.

The device of any prior embodiment, wherein at least one of the firstplate and the second plate has an area across its major surface of about400 mm².

The device of any prior embodiment, further comprising a plurality ofspherical spacers disposed between the first plate and the second plate.

The device of any prior embodiment, further comprising a plurality ofspacers having a height of about 10 um, wherein the plurality of spacersare disposed between the first plate and the second plate.

The device of any prior embodiment, wherein the at least a portion ofthe liquid sample comprises a volume of the sample along a path of theelectromagnetic radiation.

The device of any prior embodiment, further comprising a hingeconfigured to connect the first plate with the second plate, and coupledto an edge of the first plate or the second plate.

A device, comprising:

a first plate;

a second plate having a thickness less than or equal to 100 μm, whereinthe second plate is separated from the first plate in a parallelarrangement by a distance less than or equal to the thickness of thesecond plate; and

a heating/cooling layer disposed on either the first plate or the secondplate,

wherein at least a portion of a liquid sample sandwiched between thefirst plate and the second plate is cooled at a rate of at least 30°C./sec when the heating/cooling layer is not receiving electromagneticradiation generated by an optical source.

The device of any prior embodiment, further comprising a light absorbinglayer disposed on the heating/cooling layer, wherein the light absorbinglayer has an average light absorptance of at least 30%.

The device of any prior embodiment, wherein the light absorbing layercomprises black paint.

The device of any prior embodiment, wherein the first plate is movablerelative to the second plate.

The device of any prior embodiment, wherein a thickness of theheating/cooling layer is less than or equal to 3 μm.

The device of any prior embodiment, wherein at least one of the firstplate and the second plate has an area across its major surface of about400 mm².

The device of any prior embodiment, further comprising a plurality ofspherical spacers disposed between the first plate and the second plate.

The device of any prior embodiment, further comprising a plurality ofspacers having a height of about 10 um, wherein the plurality of spacersare disposed between the first plate and the second plate.

The device of any prior embodiment, wherein the at least a portion ofthe liquid sample comprises a volume of the sample that is adjacent tothe heating/cooling layer.

The device of any prior embodiment, further comprising a hingeconfigured to connect the first plate with the second plate, and coupledto an edge of the first plate or the second plate.

A device, comprising:

a first plate;

a second plate having a thickness less than or equal to 100 μm, whereinan inner surface of the second plate is separated from an inner surfaceof the first plate in a parallel arrangement by a distance less than orequal to the thickness of the second plate;

a heating/cooling layer disposed on the inner surface or on an outersurface of the second plate; and

a layer of reagents dried on the inner surface of the first plate.

The device of any prior embodiment, further comprising a light absorbinglayer disposed on the heating/cooling layer, wherein the light absorbinglayer has an average light absorptance of at least 30%.

The device of any prior embodiment, wherein the light absorbing layercomprises black paint.

The device of any prior embodiment, wherein the first plate is movablerelative to the second plate.

The device of any prior embodiment, wherein a thickness of theheating/cooling layer is less than or equal to 3 μm.

The device of any prior embodiment, wherein at least one of the firstplate and the second plate has an area across its major surface of about400 mm².

The device of any prior embodiment, further comprising a plurality ofspherical spacers disposed between the first plate and the second plate.

The device of any prior embodiment, further comprising a plurality ofspacers having a height of about 10 um, wherein the plurality of spacersare disposed between the first plate and the second plate.

The device of any prior embodiment, wherein the layer of dried reagentscomprises reagents used for nucleic acid amplification.

The device of any prior embodiment, further comprising a hingeconfigured to connect the first plate with the second plate, and coupledto an edge of the first plate or the second plate.

A system, comprising:

a device, comprising:

a first plate comprising a polymer material and having a thickness lessthan or equal to 100 μm,

a second plate comprising a polymer material and having a thickness lessthan or equal to 100 μm, wherein the second plate is separated from thefirst plate in a parallel arrangement by a distance less than or equalto the thickness of the second plate,

a heating/cooling layer disposed on either the first plate or the secondplate, the heating/cooling layer having a thickness and a thermalconductivity between 6×10⁻⁵ W/K multiplied by the thickness of theheating/cooling layer and 1.5×10⁻⁴ W/K multiplied by the thickness ofthe heating/cooling layer, and

a support frame configured to support at least one of the first plateand the second plate;

a housing having a first opening configured to receive the device and atleast one other opening;

an optical source configured to direct electromagnetic radiation towardsthe heating/cooling layer,

wherein the heating/cooling layer is configured to absorb at least aportion of the electromagnetic radiation such that at least a portion ofa liquid sample sandwiched between the first plate and the second plateis heated at a rate of at least 30° C./sec, and

wherein at least the portion of the liquid sample sandwiched between thefirst plate and the second plate is cooled at a rate of at least 30°C./sec when the heating/cooling layer is not receiving theelectromagnetic radiation generated by the optical source, and

wherein the system consumes less than 500 mW of power.

The system of any prior embodiment, wherein the device further comprisesa light absorbing layer disposed on the heating/cooling layer, whereinthe light absorbing layer has an average light absorptance of at least30%.

The system of any prior embodiment, wherein the light absorbing layercomprises black paint.

The system of any prior embodiment, wherein the first plate is movablerelative to the second plate.

The system of any prior embodiment, wherein a thickness of theheating/cooling layer is less than or equal to 3 μm.

The system of any prior embodiment, wherein at least one of the firstplate and the second plate has an area across its major surface of about400 mm².

The system of any prior embodiment, wherein the optical source comprisesa light emitting diode (LED.)

The system of any prior embodiment, further comprising an optical pipeconfigured to guide the electromagnetic radiation from the opticalsource to the heating/cooling layer.

The system of any prior embodiment, wherein the at least one otheropening of the housing is configured to be aligned over at least theportion of the liquid sample sandwiched between the first plate and thesecond plate when the device is placed within the housing via the firstopening.

The system of any prior embodiment, wherein the support frame isconfigured to support at least the first plate or the second plate alonga perimeter of the first plate or second plate.

A system, comprising:

a device, comprising:

a first plate,

a second plate having a thickness less than or equal to 100 μm, whereinthe second plate is separated from the first plate in a parallelarrangement by a distance less than or equal to the thickness of thesecond plate,

a heating/cooling layer disposed on either the first plate or the secondplate, and

a support frame configured to support at least one of the first plateand the second plate; and

an optical source configured to direct electromagnetic radiation towardsthe heating/cooling layer,

wherein at least a portion of a liquid sample sandwiched between thefirst plate and the second plate is cooled at a rate of at least 30°C./sec when the heating/cooling layer is not receiving theelectromagnetic radiation generated by the optical source.

The system of any prior embodiment, wherein the device further comprisesa light absorbing layer disposed on the heating/cooling layer, whereinthe light absorbing layer has an average light absorptance of at least30%.

The system of any prior embodiment, wherein the light absorbing layercomprises black paint.

The system of any prior embodiment, wherein the first plate is movablerelative to the second plate.

The system of any prior embodiment, wherein a thickness of theheating/cooling layer is less than or equal to 3 μm.

The system of any prior embodiment, wherein at least one of the firstplate and the second plate has an area across its major surface of about400 mm².

The system of any prior embodiment, wherein the optical source comprisesa light emitting diode (LED.)

The system of any prior embodiment, wherein the LED comprises a blueLED.

The system of any prior embodiment, further comprising an optical pipeconfigured to guide the electromagnetic radiation from the opticalsource to the heating/cooling layer.

The system of any prior embodiment, wherein the support frame isconfigured to support at least the first plate or the second plate alonga perimeter of the first plate or second plate.

A system, comprising:

a device, comprising:

a first plate,

a second plate having a thickness less than or equal to 100 μm, whereinthe second plate is separated from the first plate in a parallelarrangement by a distance less than or equal to the thickness of thesecond plate,

a heating/cooling layer disposed on either the first plate or the secondplate; and

an optical source configured to direct electromagnetic radiation towardsthe heating/cooling layer, wherein the system consumes less than 500 mWof power.

The system of any prior embodiment, wherein the device further comprisesa light absorbing layer disposed on the heating/cooling layer, whereinthe light absorbing layer has an average light absorptance of at least30%.

The system of any prior embodiment, wherein the light absorbing layercomprises black paint.

The system of any prior embodiment, wherein the first plate is movablerelative to the second plate.

The system of any prior embodiment, wherein a thickness of theheating/cooling layer is less than or equal to 3 μm.

The system of any prior embodiment, wherein at least one of the firstplate and the second plate has an area across its major surface of about400 mm².

The system of any prior embodiment, wherein the optical source comprisesa light emitting diode (LED.)

The system of any prior embodiment, wherein the LED comprises a blueLED.

The system of any prior embodiment, further comprising an optical pipeconfigured to guide the electromagnetic radiation from the opticalsource to the heating/cooling layer.

The system of any prior embodiment, further comprising a support frameconfigured to support at least the first plate or the second plate alonga perimeter of the first plate or second plate.

A system, comprising:

a device, comprising:

a first plate,

a second plate having a thickness less than or equal to 100 μm, whereinthe second plate is separated from the first plate in a parallelarrangement by a distance less than or equal to the thickness of thesecond plate,

a heating/cooling layer disposed on either the first plate or the secondplate, and

a support frame configured to support at least one of the first plateand the second plate;

a housing having a first opening configured to receive the device and atleast one other opening; and

an optical source configured to direct electromagnetic radiation throughthe at least one other opening of the housing and towards theheating/cooling layer,

wherein a liquid sample sandwiched between the first plate and thesecond plate is cooled at a rate of at least 30° C./sec when theheating/cooling layer is not receiving the electromagnetic radiationgenerated by the optical source.

The system of any prior embodiment, wherein the device further comprisesa light absorbing layer disposed on the heating/cooling layer, whereinthe light absorbing layer has an average light absorptance of at least30%.

The system of any prior embodiment, wherein the light absorbing layercomprises black paint.

The system of any prior embodiment, wherein the first plate is movablerelative to the second plate.

The system of any prior embodiment, wherein a thickness of theheating/cooling layer is less than or equal to 3 μm.

The system of any prior embodiment, wherein at least one of the firstplate and the second plate has an area across its major surface of about400 mm².

The system of any prior embodiment, wherein the optical source comprisesa light emitting diode (LED.)

The system of any prior embodiment, wherein the LED comprises a blueLED.

The system of any prior embodiment, further comprising an optical pipeconfigured to guide the electromagnetic radiation from the opticalsource to the heating/cooling layer.

The system of any prior embodiment, wherein the support frame isconfigured to support at least the first plate or the second plate alonga perimeter of the first plate or second plate.

A method of using a device, comprising:

placing a second plate over a first plate such that a fluidic sample issandwiched between the first plate and the second plate at a thicknessdetermined by one or more spacers located on at least one of the firstplate and the second plate;

activating a heat source configured to radiate electromagnetic radiationtowards a heating layer located on either the first plate or the secondplate; and

heating, using at least the heating layer, at least a portion of thefluidic sample at a rate of at least 30° C./sec.

The method of any prior embodiment, wherein the first plate or thesecond plate further comprises a light absorbing layer disposed on theheating layer, wherein the light absorbing layer has an average lightabsorptance of at least 30%.

The method of any prior embodiment, wherein the light absorbing layercomprises black paint.

The method of any prior embodiment, further comprising closing thesecond plate over the first plate using a hinge connected between thefirst plate and the second plate.

The method of any prior embodiment, wherein a thickness of the heatinglayer is less than or equal to 3 μm.

The method of any prior embodiment, wherein at least one of the firstplate and the second plate has an area across its major surface of about400 mm².

The method of any prior embodiment, wherein activating a heat sourcecomprises activating an LED to radiate light towards the heating layer.

The method of any prior embodiment, further comprising controlling anoutput of the LED based on a measured or estimated temperature of theportion of the fluidic sample.

The method of any prior embodiment, further comprising expanding theelectromagnetic radiation using a beam expander before theelectromagnetic radiation reaches the heating layer.

The method of any prior embodiment, further comprising supporting aperimeter of either the first plate or the second plate on a supportframe.

A method of using a device, comprising:

placing a second plate over the first plate such that a fluidic sampleis sandwiched between the first plate and the second plate at athickness determined by one or more spacers located on at least one ofthe first plate and the second plate;

activating, for a given time period, a heat source configured to radiateelectromagnetic radiation towards a heating/cooling layer located oneither the first plate or the second plate;

deactivating the heat source after the given time period, wherein atleast a portion of the fluidic sample cools at a rate of at least 30°C./sec after the deactivating.

The method of any prior embodiment, wherein the first plate or thesecond plate further comprises a light absorbing layer disposed on theheating/cooling layer, wherein the light absorbing layer has an averagelight absorptance of at least 30%.

The method of any prior embodiment, wherein the light absorbing layercomprises black paint.

The method of any prior embodiment, further comprising closing thesecond plate over the first plate using a hinge connected between thefirst plate and the second plate.

The method of any prior embodiment, wherein a thickness of theheating/cooling layer is less than or equal to 3 μm.

The method of any prior embodiment, wherein at least one of the firstplate and the second plate has an area across its major surface of about400 mm².

The method of any prior embodiment, wherein activating a heat sourcecomprises activating an LED to radiate light towards the heating/coolinglayer.

The method of any prior embodiment, further comprising controlling anoutput of the LED based on a measured or estimated temperature of theportion of the fluidic sample.

The method of any prior embodiment, further comprising expanding theelectromagnetic radiation using a beam expander before theelectromagnetic radiation reaches the heating layer.

The method of any prior embodiment, further comprising supporting aperimeter of either the first plate or the second plate on a supportframe.

A method of using a device, comprising:

placing a second plate over the first plate such that a fluidic sampleis sandwiched between the first plate and the second plate at athickness determined by one or more spacers located on at least one ofthe first plate and the second plate;

activating a heat source configured to radiate electromagnetic radiationtowards a heating layer located on either the first plate or the secondplate, wherein the heat source consumes less than 500 mW of power; and

heating, using at least the heating layer, at least a portion of thefluidic sample.

The method of any prior embodiment, wherein the first plate or thesecond plate further comprises a light absorbing layer disposed on theheating layer, wherein the light absorbing layer has an average lightabsorptance of at least 30%.

The method of any prior embodiment, wherein the light absorbing layercomprises black paint.

The method of any prior embodiment, further comprising closing thesecond plate over the first plate using a hinge connected between thefirst plate and the second plate.

The method of any prior embodiment, wherein a thickness of the heatinglayer is less than or equal to 3 μm.

The method of any prior embodiment, wherein at least one of the firstplate and the second plate has an area across its major surface of about400 mm².

The method of any prior embodiment, wherein activating a heat sourcecomprises activating an LED to radiate light towards the heating layer.

The method of any prior embodiment, further comprising controlling anoutput of the LED based on a measured or estimated temperature of theportion of the fluidic sample.

The method of any prior embodiment, further comprising expanding theelectromagnetic radiation using a beam expander before theelectromagnetic radiation reaches the heating layer.

The method of any prior embodiment, further comprising supporting aperimeter of either the first plate or the second plate on a supportframe.

A method of amplifying nucleic acids, comprising:

depositing a fluidic sample containing nucleic acids on a first plate ofa fluidic device;

placing a second plate over the first plate such that the fluidic sampleis sandwiched between the first plate and the second plate, whereinreagents for nucleic acid amplification are present on the inner surfaceof the second plate;

activating a heat source configured to radiate electromagnetic radiationtowards a heating layer located on either the first plate or the secondplate;

heating, using at least the heating layer, at least a portion of thefluidic sample at a rate of at least 30° C./sec; and

accumulating nucleic acid amplification products in at least the portionof the fluidic sample sandwiched between the first plate and the secondplate.

The method of any prior embodiment, wherein the first plate or thesecond plate further comprises a light absorbing layer disposed on theheating/cooling layer, wherein the light absorbing layer has an averagelight absorptance of at least 30%.

The method of any prior embodiment, wherein the light absorbing layercomprises black paint.

The method of any prior embodiment, further comprising closing thesecond plate over the first plate using a hinge connected between thefirst plate and the second plate.

The method of any prior embodiment, wherein a thickness of theheating/cooling layer is less than or equal to 3 μm.

The method of any prior embodiment, wherein at least one of the firstplate and the second plate has an area across its major surface of about400 mm².

The method of any prior embodiment, wherein activating a heat sourcecomprises activating an LED to radiate light towards the heating/coolinglayer.

The method of any prior embodiment, further comprising controlling anoutput of the LED based on a measured or estimated temperature of theportion of the fluidic sample.

The method of any prior embodiment, further comprising expanding theelectromagnetic radiation using a beam expander before theelectromagnetic radiation reaches the heating layer.

The method of any prior embodiment, further comprising supporting aperimeter of either the first plate or the second plate on a supportframe.

A method of amplifying nucleic acids, comprising:

depositing a fluidic sample containing nucleic acids on a first plate ofa fluidic device;

placing a second plate over the first plate such that the fluidic sampleis sandwiched between the first plate and the second plate, whereinreagents for nucleic acid amplification are present on the inner surfaceof the second plate;

amplifying nucleic acids in the sample by conducting one or more PCRcycles, wherein each PCR cycle comprises a denaturing step, an annealingstep, and an elongation step;

wherein one or more of the denaturing steps, the annealing step, and/orthe elongation step comprises:

activating a heat source configured to radiate electromagnetic radiationtowards a heating layer located on either the first plate or the secondplate; and

heating, using at least the heating layer, at least a portion of thefluidic sample at a rate of at least 30° C./sec.

The method of any prior embodiment, wherein the first plate or thesecond plate further comprises a light absorbing layer disposed on theheating/cooling layer, wherein the light absorbing layer has an averagelight absorptance of at least 30%.

The method of any prior embodiment, wherein the light absorbing layercomprises black paint.

The method of any prior embodiment, further comprising closing thesecond plate over the first plate using a hinge connected between thefirst plate and the second plate.

The method of any prior embodiment, wherein a thickness of theheating/cooling layer is less than or equal to 3 μm.

The method of any prior embodiment, wherein at least one of the firstplate and the second plate has an area across its major surface of about400 mm².

The method of any prior embodiment, wherein activating a heat sourcecomprises activating an LED to radiate light towards the heating/coolinglayer.

The method of any prior embodiment, further comprising controlling anoutput of the LED based on a measured or estimated temperature of theportion of the fluidic sample.

The method of any prior embodiment, further comprising expanding theelectromagnetic radiation using a beam expander before theelectromagnetic radiation reaches the heating layer.

The method of any prior embodiment, further comprising supporting aperimeter of either the first plate or the second plate on a supportframe.

A method of amplifying nucleic acids, comprising:

depositing a fluidic sample containing nucleic acids on a first plate ofa fluidic device;

placing a second plate over the first plate such that the fluidic sampleis sandwiched between the first plate and the second plate, whereinreagents for nucleic acid amplification are present on the inner surfaceof the second plate;

activating, for a given time period, a heat source configured to radiateelectromagnetic radiation towards a heating/cooling layer located oneither the first plate or the second plate;

deactivating the heat source after the given time period, wherein atleast a portion of the fluidic sample adjacent to the heating/coolinglayer cools at a rate of at least 30° C./sec after the deactivating; and

accumulating nucleic acid amplification products in at least the portionof the fluidic sample sandwiched between the first plate and the secondplate.

The method of any prior embodiment, wherein the first plate or thesecond plate further comprises a light absorbing layer disposed on theheating/cooling layer, wherein the light absorbing layer has an averagelight absorptance of at least 30%.

The method of any prior embodiment, wherein the light absorbing layercomprises black paint.

The method of any prior embodiment, further comprising closing thesecond plate over the first plate using a hinge connected between thefirst plate and the second plate.

The method of any prior embodiment, wherein a thickness of theheating/cooling layer is less than or equal to 3 μm.

The method of any prior embodiment, wherein at least one of the firstplate and the second plate has an area across its major surface of about400 mm².

The method of any prior embodiment, wherein activating a heat sourcecomprises activating an LED to radiate light towards the heating/coolinglayer.

The method of any prior embodiment, further comprising controlling anoutput of the LED based on a measured or estimated temperature of theportion of the fluidic sample.

The method of any prior embodiment, further comprising expanding theelectromagnetic radiation using a beam expander before theelectromagnetic radiation reaches the heating layer.

The method of any prior embodiment, further comprising supporting aperimeter of either the first plate or the second plate on a supportframe.

A method of amplifying nucleic acids, comprising:

depositing a fluidic sample containing nucleic acids on a first plate ofa fluidic device;

placing a second plate over the first plate such that the fluidic sampleis sandwiched between the first plate and the second plate, whereinreagents for nucleic acid amplification are present on the inner surfaceof the second plate;

amplifying nucleic acids in the sample by conducting one or more PCRcycles, wherein each PCR cycle comprises a denaturing step, an annealingstep, and an elongation step;

wherein one or more of the denaturing steps, the annealing step, and/orthe elongation step comprises:

activating a heat source configured to radiate electromagnetic radiationtowards a heating layer located on either the first plate or the secondplate; and

deactivating the heat source after the given time period, wherein atleast a portion of the fluidic sample adjacent to the heating/coolinglayer cools at a rate of at least 30° C./sec after the deactivating; and

accumulating nucleic acid amplification products in at least the portionof the fluidic sample sandwiched between the first plate and the secondplate.

The method of any prior embodiment, wherein the first plate or thesecond plate further comprises a light absorbing layer disposed on theheating/cooling layer, wherein the light absorbing layer has an averagelight absorptance of at least 30%.

The method of any prior embodiment, wherein the light absorbing layercomprises black paint.

The method of any prior embodiment, further comprising closing thesecond plate over the first plate using a hinge connected between thefirst plate and the second plate.

The method of any prior embodiment, wherein a thickness of theheating/cooling layer is less than or equal to 3 μm.

The method of any prior embodiment, wherein at least one of the firstplate and the second plate has an area across its major surface of about400 mm².

The method of any prior embodiment, wherein activating a heat sourcecomprises activating an LED to radiate light towards the heating/coolinglayer.

The method of any prior embodiment, further comprising controlling anoutput of the LED based on a measured or estimated temperature of theportion of the fluidic sample.

The method of any prior embodiment, further comprising expanding theelectromagnetic radiation using a beam expander before theelectromagnetic radiation reaches the heating layer.

The method of any prior embodiment, further comprising supporting aperimeter of either the first plate or the second plate on a supportframe.

A method of amplifying nucleic acids, comprising:

depositing a fluidic sample containing nucleic acids on a first plate ofa fluidic device;

placing a second plate over the first plate such that the fluidic sampleis sandwiched between the first plate and the second plate at athickness determined by one or more spacers located on at least one ofthe first plate and the second plate, wherein reagents for nucleic acidamplification are present on the inner surface of the second plate;

activating a heat source configured to radiate electromagnetic radiationtowards a heating layer located on either the first plate or the secondplate, wherein the heat source consumes less than 500 mW of power;

heating, using at least the heating layer, at least a portion of thefluidic sample; and

accumulating nucleic acid amplification products in at least the portionof the fluidic sample sandwiched between the first plate and the secondplate.

The method of any prior embodiment, wherein the first plate or thesecond plate further comprises a light absorbing layer disposed on theheating/cooling layer, wherein the light absorbing layer has an averagelight absorptance of at least 30%.

The method of any prior embodiment, wherein the light absorbing layercomprises black paint.

The method of any prior embodiment, further comprising closing thesecond plate over the first plate using a hinge connected between thefirst plate and the second plate.

The method of any prior embodiment, wherein a thickness of theheating/cooling layer is less than or equal to 3 μm.

The method of any prior embodiment, wherein at least one of the firstplate and the second plate has an area across its major surface of about400 mm².

The method of any prior embodiment, wherein activating a heat sourcecomprises activating an LED to radiate light towards the heating/coolinglayer.

The method of any prior embodiment, further comprising controlling anoutput of the LED based on a measured or estimated temperature of theportion of the fluidic sample.

The method of any prior embodiment, further comprising expanding theelectromagnetic radiation using a beam expander before theelectromagnetic radiation reaches the heating layer.

The method of any prior embodiment, further comprising supporting aperimeter of either the first plate or the second plate on a supportframe.

A method of amplifying nucleic acids, comprising:

depositing a fluidic sample containing nucleic acids on a first plate ofa fluidic device;

placing a second plate over the first plate such that the fluidic sampleis sandwiched between the first plate and the second plate at athickness determined by one or more spacers located on at least one ofthe first plate and the second plate, wherein reagents for nucleic acidamplification are present on the inner surface of the second plate;

amplifying nucleic acids in the sample by conducting one or more PCRcycles, wherein each PCR cycle comprises a denaturing step, an annealingstep, and an elongation step;

wherein one or more of the denaturing step, the annealing step, and/orthe elongation step comprises:

activating a heat source configured to radiate electromagnetic radiationtowards a heating layer located on either the first plate or the secondplate, wherein the heat source consumes less than 500 mW of power;

heating, using at least the heating layer, at least a portion of thefluidic sample; and

accumulating nucleic acid amplification products in at least the portionof the fluidic sample sandwiched between the first plate and the secondplate.

The method of any prior embodiment, wherein the first plate or thesecond plate further comprises a light absorbing layer disposed on theheating/cooling layer, wherein the light absorbing layer has an averagelight absorptance of at least 30%.

The method of any prior embodiment, wherein the light absorbing layercomprises black paint.

The method of any prior embodiment, further comprising closing thesecond plate over the first plate using a hinge connected between thefirst plate and the second plate.

The method of any prior embodiment, wherein a thickness of theheating/cooling layer is less than or equal to 3 μm.

The method of any prior embodiment, wherein at least one of the firstplate and the second plate has an area across its major surface of about400 mm².

The method of any prior embodiment, wherein activating a heat sourcecomprises activating an LED to radiate light towards the heating/coolinglayer.

The method of any prior embodiment, further comprising controlling anoutput of the LED based on a measured or estimated temperature of theportion of the fluidic sample.

The method of any prior embodiment, further comprising expanding theelectromagnetic radiation using a beam expander before theelectromagnetic radiation reaches the heating layer.

The method of any prior embodiment, further comprising supporting aperimeter of either the first plate or the second plate on a supportframe.

A method for detecting whether a target nucleic acid sequence is presentor absent in a sample, comprising:

depositing a fluidic sample containing nucleic acids on a first plate ofa fluidic device;

placing a second plate over the first plate such that the fluidic sampleis sandwiched between the first plate and the second plate, whereinreagents for nucleic acid amplification are present on the inner surfaceof the second plate, and wherein the reagents comprise primers that canhybridize with the target nucleic acid;

activating a heat source configured to radiate electromagnetic radiationtowards a heating layer located on either the first plate or the secondplate;

heating, using at least the heating layer, at least a portion of thefluidic sample at a rate of at least 30° C./sec; and

detecting whether the fluidic sample contains amplified product of thetarget nucleic acid sequence.

The method of any prior embodiment, wherein the first plate or thesecond plate further comprises a light absorbing layer disposed on theheating/cooling layer, wherein the light absorbing layer has an averagelight absorptance of at least 30%.

The method of any prior embodiment, wherein the light absorbing layercomprises black paint.

The method of any prior embodiment, further comprising closing thesecond plate over the first plate using a hinge connected between thefirst plate and the second plate.

The method of any prior embodiment, wherein a thickness of theheating/cooling layer is less than or equal to 3 μm.

The method of any prior embodiment, wherein at least one of the firstplate and the second plate has an area across its major surface of about400 mm².

The method of any prior embodiment, wherein activating a heat sourcecomprises activating an LED to radiate light towards the heating/coolinglayer.

The method of any prior embodiment, further comprising controlling anoutput of the LED based on a measured or estimated temperature of theportion of the fluidic sample.

The method of any prior embodiment, further comprising expanding theelectromagnetic radiation using a beam expander before theelectromagnetic radiation reaches the heating layer.

The method of any prior embodiment, further comprising supporting aperimeter of either the first plate or the second plate on a supportframe.

A method for detecting whether a target nucleic acid sequence is presentor absent in a sample, comprising:

depositing a fluidic sample containing nucleic acids on a first plate ofa fluidic device;

placing a second plate over the first plate such that the fluidic sampleis sandwiched between the first plate and the second plate, whereinreagents for nucleic acid amplification are present on the inner surfaceof the second plate, and wherein the reagents comprise primers that canhybridize with the target nucleic acid;

activating, for a given time period, a heat source configured to radiateelectromagnetic radiation towards a heating/cooling layer located oneither the first plate or the second plate;

deactivating the heat source after the given time period, wherein atleast a portion of the fluidic sample adjacent to the heating/coolinglayer cools at a rate of at least 30° C./sec after the deactivating; and

detecting whether the fluidic sample contains amplified product of thetarget nucleic acid sequence.

The method of any prior embodiment, wherein the first plate or thesecond plate further comprises a light absorbing layer disposed on theheating/cooling layer, wherein the light absorbing layer has an averagelight absorptance of at least 30%.

The method of any prior embodiment, wherein the light absorbing layercomprises black paint.

The method of any prior embodiment, further comprising closing thesecond plate over the first plate using a hinge connected between thefirst plate and the second plate.

The method of any prior embodiment, wherein a thickness of theheating/cooling layer is less than or equal to 3 μm.

The method of any prior embodiment, wherein at least one of the firstplate and the second plate has an area across its major surface of about400 mm².

The method of any prior embodiment, wherein activating a heat sourcecomprises activating an LED to radiate light towards the heating/coolinglayer.

The method of any prior embodiment, further comprising controlling anoutput of the LED based on a measured or estimated temperature of theportion of the fluidic sample.

The method of any prior embodiment, further comprising expanding theelectromagnetic radiation using a beam expander before theelectromagnetic radiation reaches the heating layer.

The method of any prior embodiment, further comprising supporting aperimeter of either the first plate or the second plate on a supportframe

A method for detecting whether a target nucleic acid sequence is presentor absent in a sample, comprising:

depositing a fluidic sample containing nucleic acids on a first plate ofa fluidic device;

placing a second plate over the first plate such that the fluidic sampleis sandwiched between the first plate and the second plate, whereinreagents for nucleic acid amplification are present on the inner surfaceof the second plate, and wherein the reagents comprise primers that canhybridize with the target nucleic acid;

activating a heat source configured to radiate electromagnetic radiationtowards a heating layer located on either the first plate or the secondplate, wherein the heat source consumes less than 500 mW of power;

heating, using at least the heating layer, at least a portion of thefluidic sample; and

detecting whether the fluidic sample contains amplified product of thetarget nucleic acid sequence.

The method of any prior embodiment, wherein the first plate or thesecond plate further comprises a light absorbing layer disposed on theheating/cooling layer, wherein the light absorbing layer has an averagelight absorptance of at least 30%.

The method of any prior embodiment, wherein the light absorbing layercomprises black paint.

The method of any prior embodiment, further comprising closing thesecond plate over the first plate using a hinge connected between thefirst plate and the second plate.

The method of any prior embodiment, wherein a thickness of theheating/cooling layer is less than or equal to 3 μm.

The method of any prior embodiment, wherein at least one of the firstplate and the second plate has an area across its major surface of about400 mm².

The method of any prior embodiment, wherein activating a heat sourcecomprises activating an LED to radiate light towards the heating/coolinglayer.

The method of any prior embodiment, further comprising controlling anoutput of the LED based on a measured or estimated temperature of theportion of the fluidic sample.

The method of any prior embodiment, further comprising expanding theelectromagnetic radiation using a beam expander before theelectromagnetic radiation reaches the heating layer.

The method of any prior embodiment, further comprising supporting aperimeter of either the first plate or the second plate on a supportframe.

A method for detecting the presence or absence of an analyte in asample, comprising:

depositing a fluidic sample on a first plate of a fluidic device;

placing a second plate over the first plate such that the fluidic sampleis sandwiched between the first plate and the second plate, whereinreagents for detection of the analyte are present on the inner surfaceof the second plate;

activating a heat source configured to radiate electromagnetic radiationtowards a heating layer located on either the first plate or the secondplate;

heating, using at least the heating layer, at least a portion of thefluidic sample at a rate of at least 30° C./sec; and

detecting whether the fluidic sample contains the analyte.

A method for detecting the presence or absence of an analyte in asample, comprising:

depositing a fluidic sample containing on a first plate of a fluidicdevice;

placing a second plate over the first plate such that the fluidic sampleis sandwiched between the first plate and the second plate, whereinreagents for detection of the analyte are present on the inner surfaceof the second plate;

activating, for a given time period, a heat source configured to radiateelectromagnetic radiation towards a heating/cooling layer located oneither the first plate or the second plate;

deactivating the heat source after the given time period, wherein atleast a portion of the fluidic sample adjacent to the heating/coolinglayer cools at a rate of at least 30° C./sec after the deactivating; and

detecting whether the fluidic sample contains the analyte.

A method for detecting the presence or absence of an analyte in asample, comprising:

depositing a fluidic sample on a first plate of a fluidic device;

placing a second plate over the first plate such that the fluidic sampleis sandwiched between the first plate and the second plate, whereinreagents for detection of the analyte are present on the inner surfaceof the second plate;

activating a heat source configured to radiate electromagnetic radiationtowards a heating layer located on either the first plate or the secondplate, wherein the heat source consumes less than 500 mW of power;

heating, using at least the heating layer, at least a portion of thefluidic sample; and

detecting whether the fluidic sample contains the analyte.

A method for diagnosing a condition in a subject, comprising:

depositing a fluidic sample from the subject on a first plate of afluidic device;

placing a second plate over the first plate such that the fluidic sampleis sandwiched between the first plate and the second plate, whereinreagents for detection of an analyte are present on the inner surface ofthe second plate; activating a heat source configured to radiateelectromagnetic radiation towards a heating layer located on either thefirst plate or the second plate;

heating, using at least the heating layer, at least a portion of thefluidic sample at a rate of at least 30° C./sec; and

detecting whether the fluidic sample contains the analyte;

wherein presence or absence of the analyte indicates that the subjecthas the condition.

A method for diagnosing a condition in a subject, comprising:

depositing a fluidic sample from the subject on a first plate of afluidic device;

placing a second plate over the first plate such that the fluidic sampleis sandwiched between the first plate and the second plate, whereinreagents for detection of the analyte are present on the inner surfaceof the second plate;

activating, for a given time period, a heat source configured to radiateelectromagnetic radiation towards a heating/cooling layer located oneither the first plate or the second plate;

deactivating the heat source after the given time period, wherein atleast a portion of the fluidic sample adjacent to the heating/coolinglayer cools at a rate of at least 30° C./sec after the deactivating; and

detecting whether the fluidic sample contains the analyte;

wherein presence or absence of the analyte indicates that the subjecthas the condition.

A method for diagnosing a condition in a subject, comprising:

depositing a fluidic sample from the subject on a first plate of afluidic device;

placing a second plate over the first plate such that the fluidic sampleis sandwiched between the first plate and the second plate, whereinreagents for detection of an analyte are present on the inner surface ofthe second plate;

activating a heat source configured to radiate electromagnetic radiationtowards a heating layer located on either the first plate or the secondplate, wherein the heat source consumes less than 500 mW of power;

heating, using at least the heating layer, at least a portion of thefluidic sample; and

detecting whether the fluidic sample contains the analyte;

wherein presence or absence of the analyte indicates that the subjecthas the condition.

A method for diagnosing a condition in a subject, comprising:

depositing a fluidic sample from the subject on a first plate of afluidic device;

placing a second plate over the first plate such that the fluidic sampleis sandwiched between the first plate and the second plate, whereinreagents for detection of an analyte are present on the inner surface ofthe second plate;

activating a heat source configured to radiate electromagnetic radiationtowards a heating layer located on either the first plate or the secondplate;

heating, using at least the heating layer, at least a portion of thefluidic sample at a rate of at least 30° C./sec;

quantifying the amount of the analyte in the fluidic sample; and

comparing the amount to a control or reference amount of the analyte;

wherein a greater or reduced amount of the analyte in the samplecompared to the control or reference amount indicates that the subjecthas the condition.

A method for diagnosing a condition in a subject, comprising:

depositing a fluidic sample from the subject on a first plate of afluidic device;

placing a second plate over the first plate such that the fluidic sampleis sandwiched between the first plate and the second plate, whereinreagents for detection of an analyte are present on the inner surface ofthe second plate;

activating, for a given time period, a heat source configured to radiateelectromagnetic radiation towards a heating/cooling layer located oneither the first plate or the second plate;

deactivating the heat source after the given time period, wherein atleast a portion of the fluidic sample adjacent to the heating/coolinglayer cools at a rate of at least 30° C./sec after the deactivating;

quantifying the amount of the analyte in the fluidic sample; and

comparing the amount to a control or reference amount of the analyte;

wherein a greater or reduced amount of the analyte in the samplecompared to the control or reference amount indicates that the subjecthas the condition.

A method for diagnosing a condition in a subject, comprising:

depositing a fluidic sample from the subject on a first plate of afluidic device;

placing a second plate over the first plate such that the fluidic sampleis sandwiched between the first plate and the second plate, whereinreagents for detection of an analyte are present on the inner surface ofthe second plate;

activating a heat source configured to radiate electromagnetic radiationtowards a heating layer located on either the first plate or the secondplate, wherein the heat source consumes less than 500 mW of power;

heating, using at least the heating layer, at least a portion of thefluidic sample; and

quantifying the amount of the analyte in the fluidic sample; and

comparing the amount to a control or reference amount of the analyte;

wherein a greater or reduced amount of the analyte in the samplecompared to the control or reference amount indicates that the subjecthas the condition.

A kit, comprising:

a device of any prior embodiment; and

a pre-mixed polymerase chain reaction medium.

The kit of any prior embodiment, wherein the pre-mixed polymerase chainreaction medium comprises: a DNA template, two primers, a DNApolymerase, deoxynucleoside triphosphates (dNTPs), a bivalent cation, amonovalent cation, and a buffer solution.

The invention claimed is:
 1. A device for fluidically isolating aportion of a sample, comprising: a first plate, a second plate, aplurality of spacers, and at least one clamp, wherein: (i) the firstplate and the second plate are configured to be movable relative to eachother into different configurations, including an open configuration anda closed configuration, wherein one or both of the two plates areflexible plates, wherein each of the two plates comprises, on itsrespective surface, a sample contact area for contacting a fluidicsample, and wherein at a closed configuration the two plates sandwich asample to be analyzed into a layer of substantially uniform thicknessthat has a thickness of 200 μm or less; (ii) the plurality of spacershas a predetermined substantially uniform height that is equal to orless than 200 microns, wherein at least one of the plurality of spacersis inside the sample contact area; (iii) the at least one clampcomprises a top ring and a bottom ring, wherein the at least one clampis configurable between: (a) a non-active mode, wherein the top ring andthe bottom ring of the at least one clamp is configured to not push thefirst plate and second plate together; and (b) an active mode, whereinthe top ring and the bottom ring of the at least one clamp areconfigured to exert a force to (i) squeeze the first plate and thesecond plate and deform an area of the flexible plates that is under acompression of the at least one clamp, and (ii) deform a portion of theplurality of spacers, wherein the portion of the plurality of spacers isunder the compression of the at least one clamp, and the at least oneclamp is configured to crush the portion of the plurality of spacersunder the compression, thereby reducing the spacing between the twoplates in the area of the flexible plates that is under the compressionof the at least one clamp, and wherein the reduction of a spacingbetween the two plates is configured to reduce or prevent a fluidic flowbetween a sample portion encircled by the rings and a sample portionoutside the rings, wherein in the open configuration, the two plates arepartially or completely separated apart, the spacing between the twoplates is not regulated by the plurality of spacers, and the at leastone clamp is in non-active mode, and the sample is deposited on one orboth of the two plates; and wherein in the closed configuration, the atleast one clamp is in an active mode, at least a part of the sampledeposited on the one or both of the plates is compressed by the twoplates to form the layer of substantially uniform thickness, wherein thetwo plates and plurality of spacers are configured to regulate theuniform thickness of the layer.
 2. The device of claim 1, wherein thefirst plate or the second plate has a thickness that is less than orequal to 100 μm.
 3. The device of claim 1, wherein each of the firstplate and the second plate has a thickness less than or equal to 100 μm.4. The device of claim 2, further comprising a heating and cooling layerdisposed on the exterior of either the first plate or the second plate,wherein the heating and cooling layer is configured to receiveelectromagnetic radiation.
 5. The device of claim 4, wherein the heatingand cooling layer disposed on the exterior of either the first plate orthe second plate, wherein the heating and cooling layer has thermalconductivity of 50 W/mK or higher, and the thermal conductivity of theheating and cooling layer times the thickness of the heating and coolinglayer is 6×10⁻⁵ W/K or higher.
 6. The device of claim 4, wherein theheating and cooling layer has thermal conductivity of 50 W/mK or higher,and the thermal conductivity of the heating and cooling layer times thethickness of the heating and cooling layer is in a range of 6×10⁻⁵ W/Kto 1.5×10⁻⁴ W/K.
 7. A device for changing the temperature of a thinfluidic sample layer with image monitoring, comprising: (i) the deviceof claim 5 or 6; (ii) a heater that heats the heating and cooling layer.8. The device of claim 1 further comprising an imager that images anarea of the sample and a portion of the two plates in the area.
 9. Thedevice of claim 1, wherein the at least one clamp comprises a pluralityof clamps comprising at least 2, at least 4, at least 8, at least 16, atleast 32, or at least 64 or more top and bottom clamp rings, wherein thenumber of top rings are equal to the number of bottom rings, where thetop rings are movable relative to the bottom rings, and when the twoplates are in the closed configuration the rings oppose each other andpinch different areas of the first plate or the second plate to producemultiple isolated reaction chambers.
 10. The device of claim 1, whereinthe plurality of spacers comprise an inter-spacer distance, wherein afourth power of the inter-spacer-distance (ISD) divided by the thickness(h) and the Young's modulus (E) of the plate (ISD⁴/(hE)) is 5×10⁶um³/GPa or less.
 11. The device of claim 1, further comprising a hingethat connects the first plate and the second plate, and is configured toallow the two plates to rotate around the hinge into differentconfigurations.
 12. The device of claim 7, further comprising PCRprimers, a polymerase, a fluorescence-quencher probe oligonucleotide,dNTPs, and a template.
 13. The device of claim 7, wherein the heatingand cooling layer thermal cycles the temperature of the sample between40° C. and 90° C.
 14. The device of claim 7, wherein the imager detectsa label in the sample on the device, wherein the label is a label forqPCR.
 15. The device of claim 8, wherein the imager images air bubble inthe sample.
 16. The device of claim 1, wherein at least one plate of thefirst and second plates has a thickness of 300 um or less.
 17. Thedevice of claim 1, wherein, in the activation mode, the clamp exerts apressure of 0.01 kg/cm² or higher on the plates.
 18. The device of claim1, wherein the top ring is on the first plate and the bottom ring is onthe second plate and are movable toward each other.
 19. The device ofclaim 1, wherein the first plate and/or second plate comprise a well,and in the closed configuration, a flow of the sample from an inside toa outside of the well during thermal cycling or temperature changing isreduced compared to without using a clamping structure.
 20. The deviceof claim 1, wherein the at least one clamp comprises a plurality ofclamps, each of the plurality of clamps reduces a flow of the samplefrom an inside to an outside of a ring area during thermal cycling ortemperature changing.
 21. The device of claim 1, wherein the top ring orbottom ring of at least one clamp has a cross-section that is round,square, triangular, or rectangular with optional rounded corners. 22.The device of claim 1, wherein the cross-section of the top ring has ashape that is different to the bottom ring.
 23. The device of claim 1,wherein in the active mode, the clamp crushes the spacers between therings, thereby creating an at least partially isolated reaction chamberin the device.
 24. The device of claim 20 wherein the ring area is lessthan 5 mm², less than 2 mm², less than 1 mm², less than 0.5 mm², or lessthan 0.1 mm².
 25. The device of claim 1, wherein the spacers arearranged on the respective plates at a surface density of greater thanone per μm², greater than one per 10 μm², greater than one per 100 μm²,greater than one per 500 μm², greater than one per 1,000 μm², greaterthan one per 5,000 μm², or in a range between any of the two values. 26.The device of claim 4, wherein the heating and cooling layer comprises aheating zone configured to receive heating energy from a heating source.27. The device of claim 26, wherein an area ratio of the heating zone tothe heating and cooling layer is 1/1000, 1/500, 1/200, 1/100, 1/50,1/20, 1/10, 1/5, 1/2, 2/3, or in a range between any two of the abovevalues.
 28. The device of claim 4, wherein the heating layer and coolinglayer comprises metallic plasmonic materials, metamaterials, blacksilicon, graphite, carbon nanotube, silicon sandwich, graphene, orsuperlattice, or a combination thereof.
 29. The device of claim 4, theheating and cooling layer comprises a cooling layer that is a thermalradiative cooling layer with a thermal conductivity and has a surfacethermal radiation capability that is at least 50% of that of ablackbody.
 30. The device of claim 29, wherein the heating zone has asurface that has an average light absorptance of 30%, 40%, 50%, 60%,70%, 80%, 90%, 95%, 100%, or in a range between any of the two values.